WO1993003736A1 - Oligonucleotides a reticulation pour la formation de triple brin a mediation enzymatique - Google Patents

Oligonucleotides a reticulation pour la formation de triple brin a mediation enzymatique Download PDF

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Publication number
WO1993003736A1
WO1993003736A1 PCT/US1992/007101 US9207101W WO9303736A1 WO 1993003736 A1 WO1993003736 A1 WO 1993003736A1 US 9207101 W US9207101 W US 9207101W WO 9303736 A1 WO9303736 A1 WO 9303736A1
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Prior art keywords
strand
odn
gene
crosslinking
target
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PCT/US1992/007101
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English (en)
Inventor
Charles R. Petrie
Rich B. Meyer, Jr.
John C. Tabone
Gerald D. Hurst
Howard B. Gamper
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Microprobe Corporation
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Priority to JP5504623A priority Critical patent/JPH06509945A/ja
Priority to EP92918930A priority patent/EP0661979A4/fr
Publication of WO1993003736A1 publication Critical patent/WO1993003736A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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

Definitions

  • This invention relates to nucleoside crosslinking agents and to the use of these compounds in the preparation of oligonucleotides. It also relates to derivatives of pyrazolo[3,4-d]pyri_nidine which are useful as nucleic acid bases for the preparation of oligonucleotides.
  • Oligonucleotides are useful as diagnostic probes for the detection of "target" DNA or RNA
  • oligonucleotide probes has been, until the present, composed of nucleic acid containing ribose or deoxyribose or, in one case, natural ⁇ -arabinose (patent publication EP 227,459).
  • a novel class of nucleotide base the 3,4- disubstituted and 3,4, 6-trisubstituted pyrazolo[3,4-d]- pyrimidines, has now been found which offers several advantages over the prior art.
  • the de novo chemical synthesis of the pyrazolopyrimidine and the resulting nucleotide allows for the incorporation of a wide range of functional groups in a variety of different positions on the nucleotide base and for the use of different sugar moieties.
  • adenine, guanine and hypoxanthine analogs are obtained from a single nucleoside precursor. Additionally, the synthesis does not require the use of toxic heavy metals or expensive catalysts.
  • crosslinkable nucleotide probes for use in therapeutic and diagnostic applications is related to the pioneering work of B.R. Baker, "Design of Active-Site-Directed Irreversible Enzyme Inhibitors,” Wiley, New York, (1967), who used what was termed
  • active-site-directed enzyme inhibitors in chemotherapeutic applications.
  • Oligonucleotides may be used as chemotherapeutic agents to control the expression of gene sequences unique to an invading organism, such as a virus, a fungus, a parasite or a bacterium.
  • an invading organism such as a virus, a fungus, a parasite or a bacterium.
  • RNA-expression in bacteria is controlled by "antisense" RNA, which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity.
  • antisense RNA which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity.
  • mRNA targets in vivo (reviewed in Green, et al., Ann. Rev. Biochem. 55: 569- 597 (1986)). Additionally, a specific mRNA amongst a large number of mRNAs can be selectively inactivated for protein synthesis by hybridization with a complementary DNA restriction fragment, which binds to the mRNA and prevents its translation into protein on ribosomes
  • oligonucleotides complementary to portions of the HIV genome are capable of inhibiting protein expression and virus replication in cell culture. Inhibition of up to 95% was obtained with oligonucleotide concentrations of about 70 ⁇ M.
  • crosslinking suggests potential problems that must be circumvented.
  • the oligonucleotide containing a crosslinking. arm might covalently bond to the target sequence so readily that mismatching of sequences will occur, possibly resulting in host toxicity.
  • the crosslinking reaction must be fast enough to occur before correctly matched sequences can dissociate.
  • European Patent Application No. 86309090.8 describes the formation of chemically modified DNA probes such as 5-substituted uridinyl in which the substituent does not crosslink but contains a chemical or physical reporter group.
  • WO8707611 describes a process for labeling DNA fragments such as by chemically modifying the fragment followed by reaction with a fluorescent dye.
  • Yabusaki et al. in U.S. Patent No. 4,599,303 disclose a scheme for covalently crosslinking oligonucleotides such as by formation of furocoumarin monoadducts of thymidine which are made to covalently bond to other nucleotides upon photoexcitation.
  • EP 0259186 describes adducts of macromolecules and biotin which can be used as cross- linking nucleic acid hybridization probes.
  • WO8503075 describes crosslinking disulfonic esters useful as nucleic acid fragmentation agents.
  • DE3310337 describes the covalent crosslinking of single-stranded polynucleo- tides to such macromolecules as proteins with the
  • probe oligonucleotides consisting of sufficient base sequences to identify target sequences with high specificity, that are provided with one or more crosslinking arms which readily form covalent bonds with specific complementary bases.
  • Such oligonucleotides may be used as highly selective probes in
  • oligonucleotides may also be5used as antisensing agents of RNAs, e.g., in chemotherapy.
  • This invention is directed to crosslinking agents which accomplish crosslinking between specific sites on adjoining strands of oligonucleotides.
  • the crosslinking reaction observed is of excellent
  • the invention is also directed to oligonucleotides comprising at least one of these crosslinking agents and to the use of the resulting novel oligonucleotides for diagnostic and therapeutic purposes.
  • the crosslinking agents of this invention are derivatives of nucleotide bases with a crosslinking arm and are of the following formula (!'):
  • R 1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q 1 ' Q 2 and Q 3 , or with a reactive precursor thereof suitable for nucleotide bond formation;
  • d is hydroxy, phosphate or diphosphate
  • Q 3 is CH 2 -R', S-R', O-R', or N-R'R";
  • each of R 1 and R" is independently hydrogen or C h alkyl
  • B is a nucleic acid base or analog thereof that ⁇ is a component of an oligonucleotide
  • Y is a functional linking group
  • each of m and q is independently 0 to 8, inclusive;
  • r is 0 or 1
  • A' is a leaving group
  • the invention also provides novel oligonucleotides comprising at least one of the above nucleotide base derivatives of formula I'.
  • Nucleotides of this invention and oligonucleotides into which the nucleotides have been incorporated may be used as probes. Since probe hybridization is reversible, albeit slow, it is desirable to ensure that each time a probe hybridizes with the correct target sequence, the probe is irreversibly attached to that sequence.
  • nucleotide bases of the present invention will permanently modify the target strand, or cause depurination.
  • the oligonucleotides of this invention are useful in the identification, isolation, localization and/or detection of complementary nucleic acid sequences of interest in cell-free and cellular systems. Therefore, the invention further provides a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucleotide probe comprising at least one of a labeled nucleotide base of the present
  • the invention further describes methods for inactivating gene function involving combination of a crosslinkable anti-gene ODN and a recombination enzyme.
  • Coating the ODN with a recombination enzyme facilitates the search for homology with in the target gene and subsequent triple strand formation.
  • Crosslinking of resultant triple strand complexes inactivates gene function.
  • a crosslinkable anti-gene nucleoprotein filament that includes (i) a nucleoside crosslinking agent covalently linked to an oligonucleotide (ODN) complementary to a target DNA sequence within a gene, and (ii) a recombination enzyme non-covalently associated with the ODN is also described.
  • This invention also provides novel substituted pyrazolo[-3,4-d]pyrimidines which are useful as a
  • nucleotide base in preparing nucleosides and nucleotides, rather than the natural purine or pyrimidine bases or the deazapurine analogs.
  • Figure 1 depicts a modified deoxyuridine residue of an oligodeoxynucleotide crosslinked via an acetamidopropyl sidearm to a deoxyguanosine residue located two sites away from the complementary base along the 5' direction.
  • Figure 2 depicts an autoradiogram of 32 P-labeled HPV target and crosslinked product following cleavage at the 3' side of the crosslinked guanosine.
  • Lane 1 :
  • Figure 3 depicts an autoradiogram of 32 P-labeled HPV target and crosslinked product showing hybrid
  • Lane 1 Control 32 P-labeled CMV target. Lane 2: 24 hour reaction at 20°C. Lane 3: 72 hour reaction at 20°C. Lane 4: 24 hour reaction at 30°C. Lane 5: 72 hour reaction at 30°C. Reaction solutions were treated with 2-aminoethanothiol, which quenches the iodoacetamido group.
  • This invention provides novel substituted nucleotide bases with a crosslinking arm which are useful in preparing nucleosides and nucleotides and are useful as crosslinking agents.
  • the substituted bases are of the following-formula (1'):
  • R 1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q 1 , Q 2 and Q 3 , or with a reactive precursor thereof suitable for nucleotide bond formation;
  • Q 1 is hydroxy, phosphate or diphosphate
  • Q 3 is CH 2 -R' S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C 1-6 alkyl;
  • B is a nucleic acid base or analog thereof that is a component of an oligonucleotide
  • Y is a functional linking group
  • each of m and q is independently 0 to 8, inclusive;
  • r is 0 or 1
  • A' is a leaving group
  • the sugar moiety or analog thereof is selected from those useful as a component of a nucleotide.
  • a moiety may be selected from, for example, ribose, deoxyribose, pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, pentofuranose, xylose, lyxose, and
  • the sugar moiety is preferably ribose, deoxyribose, arabinose or 2'-O-methylribose and embraces either anomer, ⁇ or ⁇ .
  • the phosphorus derivative attached to the sugar moiety is conveniently selected from, for example, monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, pho ⁇ phorodithioate, and the like.
  • a reactive precursor suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucleotide.
  • Phosphoruscontaining groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites. Alternatively, activated phosphate diesters may be employed for this purpose.
  • the nucleic acid base or analog thereof (B) may be chosen from the purines, the pyrimidines, the deazapurines and the pyrazolopyrimidines. It is preferably selected from uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo- [2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyri- midin-5-yl, 4-aminopyrazolo[3,4-d]pyrimidin-3-yl or 4- amino-6-oxopyrazolo[3,4-d]pyrimidin-3-yl, where the purines are attached to the sugar moiety of the
  • oligonucleotides via the 9-position the pyrimidines via the l-position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the l-position.
  • nucleophilic properties are capable of serving as a point of attachment of the -(CH 2 ) m -A' group. Amino groups and blocked derivatives thereof are preferred.
  • the leaving group A' may be chosen from, for example, such groups as chloro, bromo, iodo, SO 2 R'", or S + R'"R"", where each of R'" and R"" is independently
  • C 1-6 alkyl or aryl or R'" and R"" together form a C 1-6 - alkylene bridge. Chloro, bromo and iodo are preferred.
  • the leaving group will be altered by its leaving ability. Depending on the nature and reactivity of the particular leaving group, the group to be used is chosen in each case to give the desired specificity of the irreversibly binding probes.
  • 5-position of the pyrimidines lie in the major groove of the B-form duplex of double-stranded nucleic acids.
  • These positions can be substituted with side chains of considerable bulk without interfering with the hybrid- ization properties of the bases.
  • These side arms may be introduced either by derivatization of dThd or dCyd, or by straightforward total synthesis of the heterocyclic base, followed by glycosylation.
  • These modified nucleosides may be converted into the appropriate activated nucleotides for incorporation into oligonucleotides with an automated DNA synthesizer.
  • the crosslinking arm is attached at the 3-position, which is
  • the crosslinking side chain should be of sufficient length to reach across the major groove from a purine 7- or 8-position, pyrimidine 5-position, pyrrolopyrimidine 5-position or pyrazolopyrimidine 3-position and reacting with the N-7 of a purine (preferably
  • the side chain should be of at least three atoms, preferably of at least five atoms and more preferably of at least six atoms in length.
  • a generally preferred length of the side chain is from about 5 to about 9 carbon atoms.
  • the target strand base which is being attacked paired to the first or second base which is on the 3' side of the modified base in the oligonucleotide containing the crosslinking arm.
  • the target sequence for a probe containing a modified uracil should contain the complement GZA
  • oligonucleotide containing UZC preferably UCC
  • U d ⁇ rd 5-substituted with the crosslinking arm.
  • the adenine-modified AZ 1 C triplet would target GZ 1 T, where Z 1 is any base.
  • the first class is the 5-substituted-2'-deoxyuridines whose general structure is presented below:
  • the 5- (substituted) -2 ' -deoxyuridines may be prepared by the routes shown in Schemes 1 and 2.
  • acetylene-coupled product (XXII).
  • the acetylenic dUrd analog XXII is reduced, with Raney nickel for example, to give the saturated compound (XXIII), which is then used for direct conversion to a reagent for use on an automated DNA synthesizer, as described below.
  • the second class of modified nucleoside is a group of 2'-deoxy-4-aminopyrazolo[3,4-d]pyrimidine derivatives.
  • the general structure of these derivatives is presented below:
  • the above compounds are derived from a novel group of derivatives of 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines.
  • the 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines and their synthesis are disclosed in commonly owned, copending application Serial No. 250,474, the entire disclosure of which is incorporated herein by reference. They have the following formula (I):
  • R 1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q 1 ' Q 2 and Q 3 , or with a reactive precursor thereof suitable for nucleotide bond formation; provided that when R 3 is hydrogen, then R 1 cannot be hydrogen;
  • Q 1 is hydroxy, phosphate or diphosphate
  • Q 3 is CH 2 -R', S-R', O-R', or N-R'R"; each of R' and R" is independently hydrogen or C 1-6 alkyl;
  • R 3 is hydrogen or the group -W-(X) n -A; each of W and X is independently a chemical linker arm;.
  • A is an intercalator, a metal ion chelator, an electrophilic crosslinker, a photoactivatable crosslinker, or a reporter group;
  • each of R 4 and R 6 is independently H, OR, SR,
  • R is H or C 1-6 alkyl
  • n is zero or one
  • t is zero to twelve.
  • the sugar moiety or its analog is selected from those useful as a component of a nucleotide.
  • a moiety may be selected from, for example, pentose, deoxypentose, hexose, deoxyhexose, ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl.
  • the sugar moiety is preferably ribose, deoxyribose, arabinose or 2'-O-methylribose and embraces either anomer, ⁇ or ⁇ .
  • the phosphorus derivative attached to the sugar moiety is conveniently selected from, for example, monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, phosphorodithioate, and the like.
  • a reactive precursor suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucleotide.
  • Reactive groups particularly useful in the present invention are those containing phosphorus.
  • Phosphorusocontaining groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites.
  • activated phosphate diesters may be employed for this purpose.
  • Linker arms may include alkylene groups of 1 to 12 carbon atoms, alkenylene groups of 2 to 12 carbon atoms and 1 or 2 olefinic bonds, alkynylene groups of 2 to 12 carbon atoms and 1 or 2acetylenic bonds, or such groups substituted at a
  • Such functionalities including aliphatic or aromatic amines, exhibit nucleophilic properties and are capable of serving as a point of attachment of the functional group (A).
  • the linker arm moiety (W alone or together with X) is preferably of at least three atoms and more
  • the terminal nucleophilic group is preferably amino or chemically blocked derivatives thereof.
  • Intercalators are planar aromatic bi-, tri- or polycyclic molecules which can insert themselves between two adjacent base pairs in a double-stranded helix of nucleic acid. Intercalators have been used to cause frameshift mutations in DNA and RNA. It has also
  • tethered covalently bound via a linker arm
  • it increases the binding affinity of the oligonucleotide for its target sequence, resulting in strongly enhanced stability of the complementary sequence complex.
  • At least some of the tethered intercalators also protect the oligonucleotide against exonucleases, but not against endonucleases. See, Sun et al., Nucleric Acids Res., 15: 6149-6158 (1987); Le Doan etal., Nucleic Acids Res., 15:7749-7760 (1987).
  • tetherable intercalating agents are oxazolopyridocarbazole, acridine orange, proflavine, acriflavine and derivatives of proflavine and acridine such as 3-azido-6- (3-bromopropylamino) acridine, 3-amino-6-(3-bromopentyl- amino)acridine, and 3-methoxy-6-chloro-9-(5-hydroxy- pentylamino)acridine.
  • Oligonucleotides capable of crosslinking to the complementary sequence of target nucleic acids are valuable in chemotherapy because they increase the efficiency of inhibition of mRNA translation or gene expression control by covalent attachment of the oligonucleotide to the target sequence. This can be accomplished by cross linking agents being covalently attached to the oligonucleotide, which can then be chemically activated to form crosslinkages which can then induce chain breaks in the target complementary sequence, thus inducing irreversible damage in the sequence.
  • electrophilic crosslinking moieties include alpha-halocarbonyl
  • oligonucleotides comprising at least one nucleotide base moiety of the invention are utilized as a probe in nucleic acid assays
  • a label is attached to detect the presence of hybrid polynucleotides.
  • Such labels act as reporter groups and act as means for detecting duplex formation between the target nucleotides and their complementary oligonucleotide probes.
  • a reporter group as used herein is a group which has a physical or chemical characteristic which can be measured or detected. Detectability may be provided by such characteristics as color change, luminescence, fluorescence, or radioactivity; or it may be provided by the ability of the reporter group to serve as a ligand recognition site.
  • pyrazolopyrimidines of the present invention of formula. I where R 1 is hydrogen may be prepared by the procedures outlined below and as set forth by
  • malononitrile (III) is treated with acyl halide (II) in the presence of a base to yield acylmalononitrile (IV) , which is subsequently methylated with dimethyl sulfate or diazomethane, for example, to give the substituted methoxymethylenemalononitrile (V) .
  • acyl halide (II) is then reacted with hydrazine hydrate in boiling alcohol to give the 3-substituted-5-aminopyra- zole-4-carbonitrile (VI) , which is treated with cold concentrated sulfuric acid to give the 3-substituted-5- aminopyrazole-4-carboxamide (VII) .
  • the carboxamide (VII) may alternatively be prepared by treating cyanoacetamide (XII) with acid halide (II) to give the acylcyanoacetamide (XIII) , which is then methylated, and the resulting methoxy compound (XIV) is reacted with hydrazine hydrate.
  • VI and VII are obtained by treating the corresponding VI and VII with boiling formamide.
  • VI may be treated with dialkoxymethyl ester of a carboxylic acid, at room temperature or above room temperature, and then with ammonia to give VIII
  • VII may be treated with dialkoxymethyl ester of a carboxylic acid (without subsequent ammonia treatment) , at room temperature or above room temperature, to give compound X.
  • VI and VII may be treated with an alkyl xanthate salt such as potassium ethyl xanthate and with alkyl halide such as methyl iodide, at a temperature above room temperature, followed by oxidation by a peroxide such as m-chloroperbenzoic acid (MCPBA) and subsequent treatment with ammonia to give IX and XI, respectively, where R 6 is NH 2 .
  • an alkyl xanthate salt such as potassium ethyl xanthate
  • alkyl halide such as methyl iodide
  • the compounds of formula I may be recovered from the reaction mixture in which they are formed by established procedures.
  • the sugar may be either added to the 1- position of the pyrazole VI or VII prior to further treatment or added to the 1-position of the pyrazolo[3,4- d]pyrimidine VIII, IX, X or XI.
  • the pyrazole or pyrazolopyrimidine is treated with sodium hydride and then with the glycosyl halide of the blocked sugar.
  • Oligonucleotides of the present invention may comprise at least one and up to all of their nucleotides from the substituted pyrazolo[3,4-d]pyrimidines of
  • oligonucleotides To prepare oligonucleotides, protective groups are introduced onto the nucleosides of formula I or formula I' and the nucleosides are activated for use in the synthesis of oligonucleotides.
  • the conversion to protected, activated forms follows the procedures as described for 2'-deoxynucleosides in detail in several reviews. See, Sonveaux, Bioor ⁇ anic Chemistry. 14:274-325 (1986); Jones, in "Oligonucleotide Synthesis, a Practical Approach", M.J. Gait, Ed., IRL Press, p. 23-34 (1984).
  • the activated nucleotides are incorporated into oligonucleotides in a manner analogous to that for DNA0and RNA nucleotides, in that the correct nucleotides will be sequentially linked to form a chain of nucleotides which is complementary to a sequence of nucleotides in target DNA or RNA.
  • the nucleotides may be incorporated either enzymatically or via chemical synthesis.
  • the nucleotides may be converted to their 5'-O-dimethoxy- trityl-3'-(N,N-diisopropyl)phosphoramidite cyanoethyl ester derivatives, and incorporated into synthetic oligonucleotides following the procedures in
  • the activated nucleptides may be used directly on an automated DNA synthesizer according to the procedures and instructions of the particular synthesizer employed.
  • the oligonucleotides may be prepared on the synthesizer using the standard commercial phosphoramidite or H-phosphonate chemistries.
  • amino- pyrazolopyrimidine nucleotide triphosphates may be any amino- pyrazolopyrimidine nucleotide triphosphate.
  • the leaving group such as a haloacyl group
  • addition of an ⁇ -haloacetamide may be verified by a changed mobility of the modified compound on HPLC, corresponding to the removal of the positive charge of the amino group, and by subsequent readdition of a positive charge by reaction with 2-aminoethanethiol to give a derivative with reverse phase HPLC mobility similar to the original aminoalkyl- oligonucleotide.
  • each of the following electrophilic leaving groups were attached to an amino- propyl group on human papillomavirus (HPV) probes:
  • An oligonucleotide probe according to the invention includes at least one labeled substituted pyrazolo[3,4-d]pyrimidine nucleotide moiety of formula I and/or at least one labeled substituted nucleotide base of formula I'.
  • Probes may be labeled by any one of several methods typically used in the art. A common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 1 4 c, or 32 P labeled probes or the like. Other reporter groups include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Alternatively, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents, enzymes and enzyme substrates. Alternatively, the same components may be indirectly bonded through a ligand- antiligand complex, such as antibodies reactive with a ligand conjugated with label. The choice of label
  • Radioactive probes are typically made using commercially available nucleotides containing the desired radioactive isotope.
  • the radioactive nucleotides can be incorporated into probes, for example, by using DNA synthesizers, by nick-translation, by tailing of radioactive bases to the 3' end of probes with terminal transferase, by copying M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase in the presence of radioactive dNTP's, or by transcribing RNA from templates using RNA polymerase in the presence of radioactive rNTP's.
  • Non-radioactive probes can be labeled directly with a signal (e.g., fluorophore, chemiluminescent agent or enzyme) or labeled indirectly by conjugation with a ligand.
  • a signal e.g., fluorophore, chemiluminescent agent or enzyme
  • a ligand molecule is covalently bound to the probe. This ligand then binds to a receptor molecule which is either inherently detectable or
  • Ligands and anti- ligands may be varied widely. Where a ligand has a natural "antiligand", namely ligands such as biotin, thyroxine, and cortisol, it can be used in conjunction with its labeled, naturally occurring antiligand.
  • any haptenic or antigenic compound can be used in combination with a suitably labeled antibody.
  • a preferred labeling method utilizes biotin-labeled analogs of oligonucleotides, as disclosed in Langer et al., Proc. Natl. Acad. Sci. USA, 78: 6633-6637 (1981), which isincorporated herein by reference.
  • Enzymes of interest as reporter groups will primarily be hydrolases, particularly phosphatases, esterases, ureases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, rare earths, etc.
  • Chemiluminescers include luciferin, acridinium esters and 2,3-dihydrophthalazinediones, e.g., luminol.
  • hybridization conditions are not critical and will vary in accordance with the investigator's preferences and needs.
  • Various hybridization solutions may be employed, comprising from about 20% to about 60% volume, preferably about 30%, of a polar organic solvent.
  • a common hybridization solution employs about 30-60% v/v formamide, about 0.5 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris HCl, PIPES or HEPES, about 0.05% to 0.5% detergent, such as sodium dodecylsulfate, and between 1- 10 mM EDTA, 0.01% to 5% ficoll (about 300-500 kdal), 0.1% to 5% polyvinylpyrrolidone (about 250-500 kdal), and 0.01% to 10% bovine serum albumin.
  • unlabeled carrier nucleic acids from about 0.1 to 5 mg/ml, e.g., partially fragmented calf thymus or salmon sperm, DNA, and/or partially fragmented yeast RNA and optionally from about 0.5% to 2% wt./vol. glycine.
  • Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents, such as anionic polyacrylate or polymethylacrylate, and
  • charged saccharidic polymers such as dextran sulfate.
  • hybridization technique is not essential to the invention.
  • Hybridization techniques are generally described in "Nucleic Acid Hybridization, A Practical Approach", Hames and Higgins, Eds., IRL Press, 1985; Gall and Pardue, Proc. Natl. Acad. Sci., U.S.A.,
  • the amount of labeled probe which is present in the hybridization solution may vary widely. Generally, substantial excesses of probe over the stoichiometric amount of the target nucleic acid will be employed to enhance the rate of binding of the probe to the target DNA.
  • degrees of stringency of hybridization can be employed. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for the formation of a stable duplex.
  • the degree of stringency can be controlled by temperature, ionic strength, the inclusion of polar organic solvents, and the like. For example, temperatures employed will normally be in the range of about 20° to 80°C, usually 25° to 75oC. For probes of 15-50 nucleotides in 50% formamide, the optimal temperature range can vary from 22-65°C. With routine experimentation, one can define conditions which permit satisfactory hybridization at room temperature.
  • the stringency of hybridization is also conveniently varied by changing the ionic strength and polarity of the reactant solution through manipulation of the concentration of formamide within the range of about 20% to about 50%.
  • Treatment with ultrasound by immersion of the reaction vessel into commercially available sonication baths can oftentimes accelerate the hybridization rates.
  • the glass, plastic, or filter support to which the probe-target hybrid is attached is introduced into a wash solution typically containing similar
  • reagents e.g., sodium chloride, buffers, organic
  • solvents and detergent as provided in the hybridization solution.
  • These reagents may be at similar concentrations as the hybridization medium, but often they are at lower concentrations when more stringent washing conditions are desired.
  • the time period for which the supportis maintained in the wash solutions may vary from minutes to several hours or more. Either the hybridization or the wash medium can be stringent. After appropriate stringent washing, the correct hybridization complex may now be detected in accordance with the nature of the label.
  • the probe may be conjugated directly with the label.
  • the label is radioactive
  • the support surface with associated hybridization complex substrate is exposed to X-ray film.
  • the label is fluorescent
  • the sample is detected by first irradiating it with light of a particular wavelength. The sample absorbs this light and then emits light of a different wavelength which is picked up by a detector ("Physical Biochemistry", Freifelder, D., W.H. Freeman & Co., 1982, pp. 537-542).
  • the label is an enzyme
  • the signal generated may be a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemiluminescence.
  • alkaline phosphatase will dephosphorylate indoxyl phosphate which then will participate in a reduction reaction to convert tetrazolium salts to highly, colored and insoluble formazans.
  • a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids.
  • binding occurs through ligand and antiligand interactions as between a ligand-conjugatedProbe and an antiligand conjugated with a signal.
  • the binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • the label may also allow indirect detection of the hybridization complex.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • a signal is gener ated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label.
  • the amount of labeled probe present in the hybridization solution may vary widely, depending upon the nature of the label, the amount of the labeled probe that can reasonably bind to the cellular target nucleic acid, and the precise stringency of the hybridization medium and/or wash medium. Generally, substantial probe excesses over the stoichiometric amount of the target will be employed to enhance the rate of binding of the probe to the target nucleic acids.
  • the invention is also directed to a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucleotide probe including at least one labeled substituted nucleotide moiety of formula I and/or formula I'.
  • the method comprises the steps of:
  • An assay for identifying target nucleic acid sequences utilizing an oligonucleotide probe including at least one labeled substituted nucleotide moiety of formula I and/or formula I' and comprising the above method is contemplated for carrying out the invention.
  • Such an assay may be provided in kit form.
  • a typical kit will include a probe reagent component comprising an oligonucleotide including at least one labeled nucleotide moiety of formula I or formula I', the oligonucleotide having a sequence complementary to that of the target nucleic acids; a denaturation reagent for converting double-stranded nucleic acid to single- stranded nucleic acid; and a hybridization reaction mixture.
  • the kit can also include a signal-generating system, such as an enzyme for example, and a substrate for the system.
  • ODNs Oligodeoxynucleotides
  • sequence specific pharmaceutical agents for the inhibition of gene expression. For instance,
  • ODNs may inhibit the expression of specific gene products through formation of duplexes upon hybridization with complementary messenger RNAs (mRNAs). More specifically, these "antisense" ODNs are believed to inhibit the processing or translation of message
  • antisense ODNs may be useful as anti-viral, antiparasitic, and anti-cancer agents. Further, antisense ODNs provide a unique opportunity for rational drug development, since a genetic target offers both extraordinar specificity and universality with respect to potential target sequences.
  • antisense technology is beset with certain fundamental disadvantages.
  • One major challenge involves development of antisense ODNs with sufficient potency to warrant in vivo testing.
  • Antisense ODN formulations that exhibit nuclease resistance, rapid cellular uptake, and efficient and stable hybridization to the target RNA sequence are desirable. Improvement of one or more of these properties without concomitant de eterious effects on other properties may be difficult.
  • phosphorothioates exhibit excellent nuclease resistance relative to unmodified ODNs, but methylphosphonate ODNs form DNA-RNA hybrids that are refractile to RNase H and phosphorothioate ODNs are poorly taken up into cells.
  • moieties to ODNs in order to potentiate their antisense activity.
  • moieties that interact directly with the RNA target upon hybridization such as pendant intercalating groups that stabilize ODN-target hybrids, ODNs with freeradical-based RNA cleaving activity or ODNs capable of covalently linking to their targets upon hybridization
  • Direct cleavage or cross- linkage of an mRNA target by an ODN renders the RNAinactive.
  • receptor-mediated cellular targeting include conjugation of a cholesteryl group that may act as an lipophilic anchor for the antisense ODN; encapsulation of ODNs into liposomes; and linkage of ODNs to soluble macromolecular complexes.
  • a cholesteryl group that may act as an lipophilic anchor for the antisense ODN
  • encapsulation of ODNs into liposomes and linkage of ODNs to soluble macromolecular complexes.
  • anti-gene A variation of the "antisense” approach to rational drug design is termed "anti-gene”.
  • antisense ODNs target single stranded mRNA
  • anti-gene ODNs hybridize with and are capable of inhibiting the function of double-stranded DNA. More specifically, anti-gene ODNs form sequence-specific triple-stranded complexes with a double stranded DNA target and thus interfere with the replication or transcription of selected target genes.
  • DNA is the repository for all genetic information, including regulatory control sequences and non-expressed genes, such as dormant proviral DNA genomes.
  • anti-gene ODNs have broader applicability and are potentially more powerful than antisense ODNs that merely inhibit mRNA processing and translation.
  • Anti-gene ODNs in the nuclei of living cells can form sequence-specific complexes with chromosomal DNA.
  • the resultant triplexes can inhibit restriction and/or transcription of the target double stranded DNA.
  • anti-gene interference with DNA functioning has longer lasting effects than the corresponding antisense inhibition of mRNA function.
  • Mammalian cell DNA does not turnover; in fact, cells possess sophisticated pathways capable of repairing lesions in DNA that may arise from environmental insults or from spontaneous rearrangements. In contrast, mRNA is transient and may exist only for minutes within a cell. The constant turnover of an mRNA species and the
  • anti-sense ODNs will provide relatively short term effects. While cellular uptake of anti-gene ODNs may need to be augmented to achieve sufficient intracellular concentrations, once within the cell the ODNs naturally concentrate in the nucleus.
  • Anti-gene therapy is based on the observation that certain DNA homopolymers can form triple-stranded complexes.
  • the third strand resides in the major groove of the Watson Crick base-paired double helix, where it hydrogen bonds to one of the two parental strands.
  • a binding code governs the recognition of base pairs by a third base (see allowed triplets below). In each case, the third strand base is presented first and is followed by the base pair;
  • Cytosine/thymidine-, guanine/adenine- and guanine/thymidine-containing ODNs can sequence- specifically bind to homopurine runs in double-stranded DNA. These recognition motifs are based on Hoogstein or reverse Hoogstein base pairing. In the C/T recognition motif, the ODN is parallel to the homopurine strand of the duplex; in the G/A recognition motif, the ODN is anti-parallel to the homopurine strand; in the G/T recognition motif, the ODN may bind parallel or antiparallel to the homopurine strand of the duplex,
  • recognition motifs may be sequence dependent.
  • the sequence specificity of anti-gene ODNs using the C/T recognition motif permits hybridization of such ODNs to homopurine runs in plasmid DNA and in yeast chromosomes. Since ODN binding is restricted to homopurine runs, it would be advantageous to identify additional heterocycles that can recognize the remaining two base pairs, i.e., C- G and T-A. While guanosine may be used in the third Strand to recognize T-A base pairs, this interaction involves only one hydrogen bond and is relatively
  • anti-gene ODNs may be modified with a variety of pendant groups designedto augment their activity.
  • intercalating groups, cleaving agents, and crosslinking moieties may be
  • substitution of 5-methyl cytosine for cytosine in the third strand ODN significantly stabilizes triplexes formed with "G"-rich homopurine runs.
  • ODNs with modified backbones, such as oligonucleoside methyl-phosphonates and phosphorothioates may form triple-stranded
  • a important disadvantage of triple strand formation as discussed above is the relatively slow kinetics of triple strand formation.
  • the claimed invention overcomes this disadvantage through enzyme catalysis of triple strand formation, with recombination enzymes particularly preferred for this purpose. More significantly, enzyme-catalyzed triple strand formation provides the immense advantage of universal sequence recognition (in contrast to the A-T and G-C recognition limitation associated with non-enzyme-mediated triple strand formation).
  • DNA molecules which plays an important role in promoting genetic diversity within a species, is catalyzed by a family of enzymes in both procaryotic andeucaryotic cells. Recombination also plays an important role in DNA repair and in the immune response.
  • Homologous recombination in E. coll serves as an illustrative example of processes that occur in both procaryotic and eucaryotic cells.
  • Studies of purifiedrecombinational enzymes from E. coli in a defined cell- free system permit division of homologous recombination into three steps.
  • the "invading" single strand is circular and the target double strand is linear.
  • the first step "presynapsis", single stranded circular DNA is coated with the multifunctional protein recA in the presence of ATP.
  • the resultant nucleoprotein filament possesses a right handed helical twist composed of 18 bases per turn and 1 monomer of recA protein per 3.6 nucleotides.
  • the single- stranded circular nucleoprotein filament conducts a two- dimensional search along a linear double-stranded DNA template for homologous sequences.
  • the search concludes with homologous alignment of the two molecules in an initial complex that has no net helical interwinding (the complex is referred to as a "paranemic joint") .
  • the DNA double helix is incorporated into the filament, and in so doing, the two DNA molecules become plectonemically coiled (i.e., helically interwound).
  • the newly incorporated third strand is homologous (in sequence and polarity) to one of the parental duplex strands, and complementary to the other parental duplex strand.
  • the three strands of DNA are believed to exist as a true hydrogen-bonded triple strand having a close association with recA.
  • the plectonemically coiled complex is stable upon
  • the plectonemically coiled synaptic complex contains 18 triads per right handed helical turn. While recA is a critical part of the complex, sequence
  • the protein-free triple stranded DNA complexes are essentially resistant to single strand-specific nucleases and exhibit very high T m 's. This is likely attributable to hydrogen bonding of the so-called "third strand" to both parental strands, as well as to the highly
  • recA may catalyze the formation of a DNA triple strand complex not otherwise attainable.
  • the two positive strands have identical sequence and
  • either positive strand is capable of forming a double stranded hybrid with the negative parental strand.
  • synaptic complexes include: (1) the length of the invading single strand; (2) the extent of shared homology between the invading single strand and the target duplex (with length of homology and absence of mismatches of particular importance); (3) the position of the shared homology region with respect to the termini of the target duplex; and (4) the superhelicity, if any, of the duplex.
  • An oligomeric 50-mer should have sufficient length to permit the formation of a stable presynaptic complex with recA.
  • Type of Joint Depending upon the positional relationship of the region of shared homology to the ends (if any) of the-targeted double-stranded DNA, the
  • Proximal joints wherein the recA-stabilized triple strand complex is located at the left hand of a linear duplex, are unstable due to recA-catalyzed strand exchange.
  • the duplex strand that is homologous to the invading third strand in the triple strand complex is displaced, resulting in a new duplex containing the invading strand and a free single strand. Since strand exchange requires a free 5' end and proceeds with a 5' to3' polarity, this process readily occurs in proximal joints.
  • distal joints and medial joints lack the appropriate ends and do not readily undergo recA-catalyzed strand exchange.
  • these joints are highly stable structures. Accordingly, within the present invention, formation of recA-stabilized triple strand complexes so as to form meidal or distal joints is preferred.
  • Superhelicity may facilitate the formation of recA-stabilized triple strands. However, high efficiency triple strand
  • the present invention combines the recA- catalyzed formation of stable triple strand complexes with synthetic anti-gene ODNs. Enzyme-catalyzed triple strand formation exhibits rapid kinetics and universal DNA sequence recognition.
  • a recA-coated anti-gene ODN serves as a "guide" that seeks homology in a target double strand DNA sequence; upon recognition and bindingof this nucleoprotein filament, recA catalyzes the formation of sequence-specific triple strand complexes.
  • an anti-gene ODN is least 30-40 nucleotides in length and has a base sequence and polarity identical to either ofthe two duplex strands in the target DNA.
  • the frequency of triplex formation and the stability of the triple strand complexes following deproteinization may be directly related to the length of the anti-gene ODN.
  • Anti-gene ODNs (of the appropriate polarity) may be usedin combination with endogenous recombinatory pathways to form sequence-specific triple strand complexes with chromosomal DNA.
  • anti-gene ODNs of a variety of lengths may be complexed with a
  • the enzyme-mediated recognition motif recognizes all four base pairs, thereby allowing targeting of any double stranded DNA sequence.
  • the recA-coated, single stranded anti-sense ODN nucleoprotein filament
  • the resultant triple strand complex is stable at physiological pH.
  • presynaptic nucleoprotein filaments such as those formed between single stranded DNA and recA, that display a weak nonspecific affinity for double stranded DNA effectively reduces the homology search from a three dimensional to a two dimensional process. Furthermore, upon homologous registry with the double strand, the nucleoprotein filament will more likely produce a triple strand complex than the corresponding interaction of double strand and a naked single strand. Because of these factors, triple strand formation between a recA-coated, single stranded ODN and an homologous double strand occurs at a reaction rate that exceeds by 1 or 2 orders of magnitude the calculated rate of spontaneous renaturation of
  • the present invention involves combination of (1) an ODN that is homologous to a portion of one target DNA duplex strand and complementary to the analogous portion of the other target DNA duplex strand; and (2) enzyme-catalyzed triple strand formation to achieve inactivation of a target DNA sequence.
  • the ODN has a crosslinking moiety covalently attached thereto.
  • cross-linkage of the third strand ODN (the anti- gene ODN) to both parental duplex strands inactivates the target DNA sequence.
  • crosslinking moiety the target DNA sequence and the environment and characteristics of the target DNA
  • inactivation of the parental duplex strands may be permanent.
  • a single administration of one or more anti-gene ODNs may abolish the expression of
  • the modified target DNA no longer supports replication or transcription. Unlike all other lesions in DNA, however, this modification is not repairable. Normally, crosslinked DNA is repaired by a combination of excision repair and homologous recombination. With crosslinked triple strand complexes, however, there will be no undamaged copies of the targeted gene to
  • the eucaryotic cell may attempt to use a misrepair (or SOS) pathway wherein the crosslink will be removed, but at the expense of mutagenesis. In such case, gene function would be irreversibly silenced by the resultant mutations.
  • misrepair or SOS
  • anti-gene ODN is combined with a recombination enzyme (for instance, in a nucleoprotein complex).
  • suitable target DNA sequences include structural genes and both up-stream and down-stream regulatory control sequences. These regulatory sequences may be involved in either
  • the anti-gene ODN will be determined and designed according to the target DNA sequence chosen for alteration of function, and will have a sequence complementary to one of the two strands of the chosen target DNA.
  • Crosslinkers suitable for use within the invention include photochemical agents, such as
  • psoralens and chemical crosslinking agents.
  • Preferred chemical crosslinking agents include those described in Section A., above; electrophilic moieties attached to the 3' and/or 5' termini of the ODN; and masked electrophilic moieties attached to the ODN.
  • Photo-crosslinking agents may be useful for topical or extracorporeal applications, for targets accessible to light exposure, as well as for in vitro use. Chemical crosslinkers may be used without limitation and are particularly preferred for use within the claimed invention.
  • Preferred recombination enzymes include
  • procaryotic and eucaryotic recombination enzymes such as recA, human recombinase and Drosophila recombinase, with human recombinase particularly preferred.
  • an anti-gene ODN is administered to a cell or a host, and upon entry to a target cell nucleus, the anti-gene ODN combines with recombination enzymes present within the nucleus.
  • the anti-gene ODN and recombination enzyme are combined ex vivo and then administered to a cell or a host as a nucleoprotein filament. In this embodiment, it may be advantageous to administer the nucleoprotein filament in a liposome.
  • RT means room temperature
  • Thin layer chromatography was performed on silica gel 60 F 254 plates (Analtech) using the followingsolvent mixtures: A- 90% methylene chloride: 10% methanol; B- 50% ethyl acetate:50% hexanes; C- 70% ethyl acetate: 10% methanol: 10% water:10% acetone; D- 50% ether:50% hexanes. Flash chromatography was performed using 60 F 254 silica (Merck). Oligonucleotides were synthesized onan Applied Biosystems Model 380B Synthesizer. Oligonucleotides were isotopically labeled using T4
  • 6-Aminocaproic acid (26 g, 0.2 mole) was dissolved in dichloromethane (200 mL) by the addition of triethylamine (100 mL). Trityl chloride (120 g, 0.45 mole) was added and the solution stirred for 36 hr. The resulting solution was extracted with 1NHCl and the organic layer evaporated to dryness. The residue was suspended in 2-propanol/1N NaOH (300 mL/100 mL) and refluxed for 3 hr. The solution was evaporated to a thick syrup and added to dichloromethane (500 mL). Water was added and acidified. The phases were separated, and the organic layer dried over sodium sulfate and evaporated to dryness. The residue was suspended in hot 2- propanol, cooled, and filtered to give 43.5 g (58%) of 6- (tritylamino) caproic acid, useful as an intermediate compound.
  • EXAMPLE 2 6-Aminocaproic acid
  • the dichloromethane solution was washed with ice cold 2N HCl (300 mL) and the biphasic mixture was filtered to remove product that precipitated (13.2 g). The phases were separated and the organic layer dried and evaporated to a thick syrup. The syrup was covered with dichloromethane and on standing deposited fine crystals of product. The crystals were filtered and dried to give 6.3 g for a total yield of 19.5 g (87%) of the product, which is useful as an intermediate.
  • Example 4 (3.5 g, 8 mmole) was treated with sodium hydride and stirred for 30 min at 0-4°C. 1-Chloro-1,2- dideoxy-3,5-di-O-toluoylribofuranose was added and the solution stirred for 1 hr at 0-4°C. The solution was poured into a saturated solution of sodium bicarbonate and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated to dryness. The residue was flash chromatographed on silica gel using toluene/ethyl acetate (5/1) as eluent. Two major
  • N-1 and N-2 isomers were isolated and identified as the N-1 and N-2 isomers in 57% (3.6 g) and 20% (1.2 g) N-1 and N-2 yields, respectively. Approximately 1 g of a mixture of N-1 and N-2 isomers was also collected. Overall yield of glycosylated material was 5.8 g (92%).
  • the triphosphate of Example 9 was incorporated into pHPV-16 using the nick tanslation protocol of Langer et al. (supra).
  • the probe prepared with the triphosphate of Example 9 was compared with probe prepared using commercially available bio-ll-dUTP (Sigma Chemical Co). No significant differences could be observed in both a filter hybridization and in in situ smears.
  • DNA polymerase 1 (U.S. Biochemicals) - 8
  • pHPV - 16 - 2.16 mg/mL which is a
  • Nucleic acid was isolated by ethanol precipitation and hybridized to pHPV-16 slotted onto nitrocellulose.
  • the hybridized biotinylated probe was visualized by a streptavidin-alkaline phosphatase conjugate with BCIP/NBT substrate.
  • Probe prepared using either biotinylated nucleotide gave identical signals.
  • the probes were also tested in an in situ format on cervical smears and showed no qualitative differences in signal and background.
  • 5-Iodo-2'-deoxyuridine (354 mg, 1 mmol) was dissolved in 10 mL of dimethylformamide. Cuprous iodide (76 mg, 0.4 mmol), tetrakis(triphenylphosphine)palladium(0) (230 mg, 0.2 mmol), and triethylamine (200 mg, 2.0 mmol) were added. 4-Phthalimidobut-1-yne (300 mg, 1.5 mmol) was added all at once and the reaction kept at 60°C for three hours. The clear yellow reaction was then evaporated and methylene chloride was added. Scratching of the flask induced crystallization of nearly all of the product which was filtered and recrystallized from 95% ethanol to give 335 mg (78%) of title compound as fine, feathery needles.
  • Nucleosides were 5'-dimethoxytritylated, following known procedures, to give around 85% yield, and the 3'-phosphoramidite was made using diisopropylamino ⁇ - cyanoethylchlorophosphite (as described in
  • Oligonucleotides were removed from the DNA synthesizer in tritylated form and deblocked using 30% ammonia at 55°C for 6 hours. Ten ⁇ L of 0.5M sodium bicarbonate was added to prevent acidification duringconcentration. The oligonucleotide was evaporated to dryness under vacuum and redissolved in 1.0 mL water.
  • oligonucleotides were purified by HPLC using 15-55% acetonitrile in 0.1N triethylammonium acetate over 20 minutes. Unsubstituted oligonucleotides came off at 10 minutes; amino derivatives took 11-12 minutes. The desired oligonucleotide was collected and evaporated to dryness, then it was redissolved in 80% aqueous acetic acid for 90 minutes to remove the trityl group.
  • nucleoside 5-(3-trifluoroacetamidoprop-1-yl)-2'-deoxyuridine was converted to the 5'-O-dimethoxytrityl-3'-(N,N- diisopropyl) phosphoramidite cyanoethyl ester derivative. This was added to a DNA synthesizer and the following 14- mer oligonucleotide sequence was prepared:
  • a corresponding 14-mer oligonucleotide was also prepared where U 1 is the unmodified deoxyuridine.
  • B (Dupont) eluted with a 20 minute gradient of 60% to 80% B composed of: A (20% acetonitrile: 80% 0.02 N NaH 2 PO 4 ) and B (1.2 N NaCl in 20% acetonitrile: 80% 0.02 N NaH 2 PO 4 ).
  • Oligo A and oligo B, as well as the above 14- mer where U 1 is the unmodified deoxyuridine were resolved in the Zorbax column, all of identical sequence, with the following retention times: unmodified 14-mer, 9.31 min; aminopropyl 14-mer (oligo A), 7.36 min; and iodoacetamidopropyl 14-mer (oligo B), 10.09 min.
  • aminobutyl 14-mer (oligo C, Example 23) was reacted with either N-hydroxysuccinimide ⁇ -iodo- acetate or N-hydroxysuccinimide 4-bromobutyrate to give the 14-mer where U 1 is 5-(4-iodoacetamidobut-1-yl)-2'- deoxyuridine or 5-(4-(4-bromobutyramido)but-1-yl)-2'- deoxyuridine, respectively.
  • the reaction of crosslinking a DNA probe to a target nucleic acid sequence contained 1 ⁇ g of haloacyl- amidoalkyl probe and 10 ng of 32 P-labeled cordycepintailed target in 200 ⁇ L of 0.1 M Tris, pH 8.0, and 0.9 M NaCl incubated at 20° or 30°C. Aliquots were removed at 24- or 72-hour intervals and diluted in 20 ⁇ L of 10 mM cysteamine to quench the haloacylamido group. These0solutions were stored at RT, and 1 ⁇ L was used for analysis by denaturing polyacrylamide gel electrophoresis (PAGE).
  • PAGE denaturing polyacrylamide gel electrophoresis
  • the target for HPV is a 30-mer, and for CMV it is a 24-mer.
  • the crosslinking probes were a 14-mer for HPV and two 15-mers for CMV. Each probe contained a single modified deoxyuridine designated as U in the sequences above.
  • Example 25 the crosslinked HPV hybrid of Example 25 (where U is 5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine) was subjected to a 10% piperidine solution at 90°C for 60 minutes. As shown by Maxam et al. (Proc. Natl. Acad.
  • Psoralen-modified, photo-crosslinkable ODNs were prepared using a photochemical procedure (REF??). Briefly, ODN was hybridized to a complementary DNA sequence and irradiated at 360 nm in the presence of 4'-hydroxymethy-4,5',8-trimethylpsoralen (HMT), thereby forming a sequence-specific, interstrand crosslink at a unique 5'-TpA-3' sequence.
  • HMT 4'-hydroxymethy-4,5',8-trimethylpsoralen
  • the HMT furan-side monoadducted ODN was isolated by denaturing PAGE.
  • a psoralen derivative to an ODN may be accomplished by chemical conjugation.
  • These synthetic schemes may be used to obtain ODNs that are coupled to a 4,5',8-trimethyl- psoralen (TMP) via a linker arm that spans the 5' terminus of the ODN and the 4' position of the psoralen.
  • TMP 4,5',8-trimethyl- psoralen
  • a modified TMP is reacted with a suitably 5'-activated, deblocked ODN (B.L. Lee et al., Biochem. 27: 3197-3202, 1988).
  • a TMP phosphoramidite linker compound is added in the last cycle of solid phase synthesis (U. Pieles and U.
  • TMP is modified at its 4' position with linker which is attached to the C8 position of deoxyadenosine.
  • a prototype, psoralenated ODN that can efficiently crosslink to both Watson and Crick strands when complexed to homologous DNA in a recA-stabilized triple strand is employed.
  • the ODN 5'-modified with a psoralenated tail is synthesized so as to optimize photo- crosslinkage within a 5'-TpA-3' sequence flanking the recA-stabilized triplex.
  • a psoralenated ODN in the presence of a recombination enzyme is used to
  • the ODN which Hoogstein base pairs to the DNA target, is modified with a 5-hydroxypsoralen moiety or a 4,5',8-trimethylpsoralen moiety at its 5' terminus.
  • the furocourmain is able to readily intercalate into a double stranded 5'-TpA-3' sequence immediately adjacent to the triple strand. Both the furan and pyrone rings are properly positioned to photoreact with thymidines.
  • psoralenated tail may be altered to obtain optimal intercalation of the psoralen into the duplex-triple strand junction.

Abstract

L'invention se rapporte à la réticulation entre des sites spécifiques sur des oligonucléotides contigus ou des oligodésoxynucléotides dans lesquels les monomères nucléosides utilisés pour effectuer la réticulation sont des pyrazolo[3,4-d] pyrimidine ribosides ou des 2'-désoxyribosides (substitués par le substituant d'alkylation). La réticulation est aidée par la présence dans les hôtes mammifères d'une enzyme de recombinaison telle que RecA. On pense que la réticulation d'acides nucléiques à double ou triple brin présente une utilité dans l'inhibition de l'expression des acides nucléiques ciblés in vivo et également comme outil de diagnostic.
PCT/US1992/007101 1991-08-19 1992-08-19 Oligonucleotides a reticulation pour la formation de triple brin a mediation enzymatique WO1993003736A1 (fr)

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WO1994024144A2 (fr) * 1993-04-19 1994-10-27 Gilead Sciences, Inc. Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
EP0635023A1 (fr) * 1992-03-05 1995-01-25 Isis Pharmaceuticals, Inc. Oligonucleotides reticules de maniere covalente
WO1996040711A1 (fr) * 1995-06-07 1996-12-19 Microprobe Corporation Oligonucleotides de reticulation
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5659022A (en) * 1996-01-05 1997-08-19 Epoch Pharmaceuticals, Inc. Oligonucleotide-cyclopropapyrroloindole conjugates as sequence specific hybridization and crosslinking agents for nucleic acids
US5763240A (en) * 1992-04-24 1998-06-09 Sri International In vivo homologous sequence targeting in eukaryotic cells
US5824796A (en) * 1988-09-28 1998-10-20 Epoch Pharmaceuticals, Inc. Cross-linking oligonucleotides
US5912340A (en) * 1995-10-04 1999-06-15 Epoch Pharmaceuticals, Inc. Selective binding complementary oligonucleotides
US5948653A (en) * 1997-03-21 1999-09-07 Pati; Sushma Sequence alterations using homologous recombination
US5955590A (en) * 1996-07-15 1999-09-21 Worcester Foundation For Biomedical Research Conjugates of minor groove DNA binders with antisense oligonucleotides
US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
US6312925B1 (en) 1997-05-08 2001-11-06 Epoch Pharmaceuticals, Inc. Methods and compositions to facilitate D-loop formation by oligonucleotides
US6312894B1 (en) 1995-04-03 2001-11-06 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
USRE38416E1 (en) 1988-09-28 2004-02-03 Epoch Biosciences, Inc. Cross-linking oligonucleotides
US7205105B2 (en) 1999-12-08 2007-04-17 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for PCR analysis
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7759126B2 (en) 2003-10-28 2010-07-20 Elitech Holding B.V. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for amplification (or PCR) analysis
US7794945B2 (en) 1995-04-03 2010-09-14 Elitech Holding B.V. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

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US5824796A (en) * 1988-09-28 1998-10-20 Epoch Pharmaceuticals, Inc. Cross-linking oligonucleotides
US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
EP0635023A1 (fr) * 1992-03-05 1995-01-25 Isis Pharmaceuticals, Inc. Oligonucleotides reticules de maniere covalente
EP0635023A4 (fr) * 1992-03-05 1996-03-20 Isis Pharmaceuticals Inc Oligonucleotides reticules de maniere covalente.
US5763240A (en) * 1992-04-24 1998-06-09 Sri International In vivo homologous sequence targeting in eukaryotic cells
WO1994017092A1 (fr) * 1993-01-26 1994-08-04 Microprobe Corporation Oligonucleotides a reticulation bifonctionnelle conçus pour se lier a une sequence de genes souhaitee d'un organisme ou d'une cellule envahisseurs
WO1994024144A3 (fr) * 1993-04-19 1995-03-16 Gilead Sciences Inc Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
WO1994024144A2 (fr) * 1993-04-19 1994-10-27 Gilead Sciences, Inc. Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
US6312894B1 (en) 1995-04-03 2001-11-06 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US7794945B2 (en) 1995-04-03 2010-09-14 Elitech Holding B.V. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US7556923B1 (en) 1995-04-03 2009-07-07 Epoch Biosciences, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US6884584B2 (en) 1995-04-03 2005-04-26 Epoch Biosciences, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US6492346B1 (en) 1995-04-03 2002-12-10 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
WO1996040711A1 (fr) * 1995-06-07 1996-12-19 Microprobe Corporation Oligonucleotides de reticulation
US5935830A (en) * 1995-06-07 1999-08-10 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
AU709924B2 (en) * 1995-06-07 1999-09-09 Epoch Pharmaceuticals, Inc. Cross-linking oligonucleotides
US5912340A (en) * 1995-10-04 1999-06-15 Epoch Pharmaceuticals, Inc. Selective binding complementary oligonucleotides
US5659022A (en) * 1996-01-05 1997-08-19 Epoch Pharmaceuticals, Inc. Oligonucleotide-cyclopropapyrroloindole conjugates as sequence specific hybridization and crosslinking agents for nucleic acids
US5955590A (en) * 1996-07-15 1999-09-21 Worcester Foundation For Biomedical Research Conjugates of minor groove DNA binders with antisense oligonucleotides
US6200812B1 (en) 1997-03-21 2001-03-13 Sri International Sequence alterations using homologous recombination
US6074853A (en) * 1997-03-21 2000-06-13 Sri Sequence alterations using homologous recombination
US5948653A (en) * 1997-03-21 1999-09-07 Pati; Sushma Sequence alterations using homologous recombination
US6312925B1 (en) 1997-05-08 2001-11-06 Epoch Pharmaceuticals, Inc. Methods and compositions to facilitate D-loop formation by oligonucleotides
US7205105B2 (en) 1999-12-08 2007-04-17 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for PCR analysis
US7485442B2 (en) 1999-12-08 2009-02-03 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5'-minor groove binder-containing probes for PCR analysis
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7718374B2 (en) 2003-10-02 2010-05-18 Elitech Holding B.V. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7759126B2 (en) 2003-10-28 2010-07-20 Elitech Holding B.V. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for amplification (or PCR) analysis

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