EP0661979A1 - 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

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Publication number
EP0661979A1
EP0661979A1 EP92918930A EP92918930A EP0661979A1 EP 0661979 A1 EP0661979 A1 EP 0661979A1 EP 92918930 A EP92918930 A EP 92918930A EP 92918930 A EP92918930 A EP 92918930A EP 0661979 A1 EP0661979 A1 EP 0661979A1
Authority
EP
European Patent Office
Prior art keywords
strand
odn
target
gene
crosslinking
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92918930A
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German (de)
English (en)
Other versions
EP0661979A4 (fr
Inventor
Charles R. Petrie
Rich B. Meyer, Jr.
John C. Tabone
Gerald D. Hurst
Howard B. Gamper
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Nanogen Inc
Original Assignee
MicroProbe Corp
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Publication date
Application filed by MicroProbe Corp filed Critical MicroProbe Corp
Publication of EP0661979A1 publication Critical patent/EP0661979A1/fr
Publication of EP0661979A4 publication Critical patent/EP0661979A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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 cross ⁇ linking 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 oligonucleo ⁇ tides.
  • Oligonucleotides are useful as diagnostic probes for the detection of "target" DNA or RNA sequences.
  • probes were made up of sequences of nucleic acid containing purine, pyrimidine or 7-deazapurine nucleotide bases (U.S. Patent 4,711,955; Robins et al., J. Can. J. Chem. , .60:554 (1982); Robins et a-I.'.-'J. Org. ' cHe . ,' 48:1854 (1983)).
  • the method for attaching chemical moieties to these bases has been via an acetoxy-mercuration reaction, which introduces covalently bound mercury atoms into the 5-position of the pyrimidine ring, the C-8 position of the purine ring or the C-7 position of a 7-deazapurine ring (Dale et al., Proc. Natl. Acad. Sci. USA f 7_0:2238 (1973); Dale et al. , Biochemistry. .14:2447 (1975)), or by the reaction of organomercurial compounds with olefinic compounds in the presence of palladium catalysts (Ruth et al., J. Org. Chem..
  • oligonucleotide probes The sugar component of oligonucleotide probes has been, until the present, composed of nucleic acid containing ribose or deoxyribose or, in one case, natural / 3-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. Similar pyrazolo[3,4-d]pyrimidines are known (Kobayashi, Chem. Pharm. Bull. , 2.1:941 (1973)); however, the substituents on the group are different from those of the present invention and their only use is as xanthine oxidase inhibitors.
  • crosslinkable nucleotide probes for use in therapeutic and diagnostic applications is related--t-o-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- ⁇ ite-directed enzyme inhibitors" in chemothera-plastic applications.
  • Oligonucleotides may be used as chemothera-plastic 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.
  • some RNA- expression in bacteria is controlled by "antisense" RNA, 0 which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity.
  • antisense RNAs__int-o eukaryotic cells have shown that they
  • 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. Importantly, they showed with labeled phosphate studies that the oligonucleotides enter cells intact and are reasonably stable to metabolism.
  • Another uncharged methylphosphonate oligonucleotide analog, an 8-nucleotide sequence complementary to the acceptor splice junction of a mRNA of—_ ⁇ erpe_j— simpiex virus, Type l can inhibit virus replication in intact Vero cells. However, fairly high concentrations (>25 mM) of this nonionic probe were required for this inhibition.
  • 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, pos ⁇ sibly resulting in host toxicity.
  • the crosslinking reaction must be fast enough to occur before correctly matched sequences can dissociate.
  • T is issue can be addressed by constructing an oligonucleotide that, upon hybridization, results in a duplex whose T B is just above the physiological temperature of 37°C.
  • the optimization can be accomplished by judicious choice of oligonucleotide length and base composition, as well as position of the modified base within the probe.
  • the probe must be long enough, however, to insure specific targeting of a unique site.
  • 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 label ⁇ ing 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 resulting complex subsequently used as a marker in hybridization experiments in the search for complementary sequences in foreign polynucleotides.
  • oligonucleotides con ⁇ sisting 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 oligonuc ⁇ leotides may be used as highly selective probes in hybr-idi_r_-ti-o ⁇ -'-__- ⁇ ays.
  • the oligonucleotides may also be 5used as antisensing agents of RNAs, e.g., in chemo ⁇ therapy.
  • 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 specificity.
  • the invention is also directed to oligo ⁇ nucleotides comprising at least one of these crosslinking agents and to the use of the resulting novel oligonucleo ⁇ tides for diagnostic and therapeutic purposes. /03736
  • crosslinking agents of this invention are derivatives of nucleotide bases with a crosslinking arm and are of the following formula (!'):
  • 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 alky1;
  • 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;
  • the invention also provides novel oligonucleo ⁇ tides comprising at least one of the above nucleotide base derivatives of formula I' .
  • Nucleotides of this invention and oligonucleo- tides 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.
  • the covalent crosslinking arm of the nucleotide bases of the present invention will permanent ⁇ ly 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 invention.
  • the invention further describes methods or 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.
  • ODN oligonucleotide
  • This invention also provides novel substituted pyrazolo ⁇ -3-r4-d. ⁇ yrimidines 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 aceta idopropyl sidear 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 /03736
  • Lane 1 32 P-labeled 15-mer size marker. 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. Reactions were quenched with 2-aminoethanothiol and treated with piperidine solution to effect cleavage.
  • Figure 3 depicts an autoradiogram of 32 P-labeled HPV target and crosslinked product showing hybrid separation by denaturing polyacrylamide gel electrophor- esis.
  • 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.
  • Crosslinking oligonucleotides 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 fpllowing—form- ⁇ ia (I*) :
  • R ⁇ is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5 1 position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q x , Q 2 and Q 3/ or with a reactive precursor thereof suitable for nucleotide bond formation;
  • Qi is hydroxy, phosphate or diphosphate;
  • Q 3 is CH 2 -R ⁇ S-R', O-R 1 , or N-R'R"; 3/03736
  • each of R' and R" is independently hydrogen or C__. 6 al_cyl;
  • 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; and A' is a leaving group.
  • the sugar moiety or analog thereof is selected from those useful as a component of a nucleotide. Such a moiety may be selected from, for example, ribose, deoxyribose, pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl.
  • the sugar moiety is preferably ribose, deoxyribose, arabinose or 2 '-O-methylribose and embraces either ano er, ⁇ or ⁇ .
  • the phosphorus derivative attached to the sugar moiety is conveniently selected from, for example, mono- phosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, pho ⁇ phorodithioate, and the like.
  • A—re-a-etive precursor suitable for internucleo- tide 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.
  • Phosphorus- containing groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphora idites. 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 deaza- purines and the pyrazolopyrimidines. It is preferably selected from uracil-5-yl, cytosin-5-yl, adenin-7-yl, /03736
  • the functional linking group Y may be chosen 10 from nucleophilic groups such as oxy, thio, amino or chemically blocked derivatives thereof, for example trifluoroaceta ido, phthalimido, CONR' , NR'CO, and S0 2 NR', where R 1
  • nucleophilic groups such as oxy, thio, amino or chemically blocked derivatives thereof, for example trifluoroaceta ido, phthalimido, CONR' , NR'CO, and S0 2 NR', where R 1
  • Such functionalities including aliphatic or aromatic amines, exhibit 15nucleophilic properties and are capable of serving as a point of attachment of the -(CH ⁇ -A 1 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, S0 2 R MI , or o S*R'"R"", where each of R'" and R"" is independently Ci.galkyl or aryl or R 1 " and R"" together form a C X . B - alkylene bridge. Chloro, bromo and iodo are preferred.
  • the leaving group will be altered by its leaving ability. ep.end___ng_o_ ⁇ ,;__-_e- nature and reactivity of the particular 5 leaving group, the group to be used is chosen in each case to give the desired specificity of the irreversibly binding probes.
  • the crosslinking side chain should be of sufficient length to reach across the major groove from a 0purine 7- or 8-position, pyrimidine 5-position, pyrrolo- pyrimidine 5-position or pyrazolopyrimidine 3-position and reacting with the N-l of a purine (preferably guanine) located above (on the oligomer 3 '-side) the base pair containing the modified analog.
  • a purine preferably guanine located above (on the oligomer 3 '-side) the base pair containing the modified analog.
  • 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 sequence for a probe containing a modified uracil should contain the complement GZA (preferably GGA) , where Z is any base, with the probe oligonucleotide containing UZC (preferably UCC) , where U is d ⁇ rd 5-substituted with the crosslinking arm.
  • GZA preferably GGA
  • UZC preferably UCC
  • the first class o is the 5-substituted-2'-deoxyuridines whose general structure is presented below:
  • the 5- (substituted) -2 ' -deoxyuridines may be prepared""by- ⁇ _:_ ⁇ _n:outes shown in Schemes 1 and 2.
  • ___ove 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-di ⁇ substituted and 3,4,6-trisubstituted pyrazolo[3,4-d]pyri- midines 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) :
  • Rj 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 l f 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-*. cannot be hydrogen;
  • Q x 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 j .galkyl;
  • 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 cross ⁇ linker, or a reporter group; each of R ⁇ and R 6 is independently H, OR, SR,
  • R is H or Cj. ⁇ alkyl n is zero or one; and _.t ,3s.zero to twelve.
  • the synthesis of 3,4-disubstituted and 3,4,6- trisubstituted pyrazolo[3,4-d]pyrimidine nucleosides and their use as reagents for incorporation into nucleic acids either enzymatically or via chemical synthesis offers several advantages over current procedures.
  • the de novo chemical synthesis of the nucleotide allows for the incorporation of a wide range of functional groups (e.g., NH 2 , SH, OH, halogen, COOH, CN, CONH 2 ) and the use of different sugar moieties.
  • adenine, guanine, and hypoxanthine analogs are obtained from a single nucleo- side precursor. And, the synthesis does not require the use of toxic heavy metals or expensive catalysts. /03736
  • 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, 5 hexose, deoxyhexose, ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclo- pentyl.
  • the sugar moiety is preferably ribose, deoxy ⁇ ribose, arabinose or 2'-O-methylribose and embraces either anomer, ⁇ or ⁇ .
  • the phosphorus derivative attached to the sugar moiety is conveniently selected from, for example, mono- phosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphonate, phosphorothioate, phosphorodithioate, and the like.
  • a reactive precursor suitable for internucleo- tide 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.
  • Phosphorus- ocontaining groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites, alkyl phosphites or alkylphosphoramidites. Alternatively, activated phosphate diesters may be employed for this purpose..
  • a chemical linker arm is 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 2 acetylenic bonds, or such groups substituted at a terminal point with nucleophilic groups such as oxy, thio, amino or chemically blocked derivatives thereof 3/03736
  • Such functionalities including aliphatic or aromatic amines, exhibit nucleo ⁇ philic 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 preferably of at least five atoms.
  • the terminal nucleo ⁇ philic 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 recently been shown that when an intercalator is covalently bound via a linker arm ("tethered") to the end of a deoxyoligonucleotide, it increases the binding affinity of the oligonucleotide for its target sequence, resulting in strongly enhanced stability of the comple ⁇ mentary sequence complex. At least some of the tethered intercalators also protect the oligonucleotide against exonucleases, but not against endonucleases.
  • tetherable intercalating agents are oxazolopyridocar- apelole, 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-chl ⁇ ro-9-(5-hydroxy- pentylamino)acridine.
  • Oligonucleotides capable of crosslinking to the complementary sequence of target nucleic acids are valu ⁇ able 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- /03736
  • linking agents being covalently attached to the oligonuc ⁇ leotide, 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 compounds, 2-chloroethylamines and epoxides.
  • 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.
  • the pyrazolopyrimidines of the present inven ⁇ tion of f.ormul______-where Rj is hydrogen may be prepared by the procedures outlined below and as set forth by Kobayashi in Chem. Pharm. Bull., £1:941-951 (1973), the disclosure of which is incorporated herein by reference.
  • alononitrile (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 diazo ethane, for example, to give the substituted methoxymethylenemalononitrile (V) .
  • acyl halide (II) is subsequently methylated with dimethyl sulfate or diazo ethane, for example, to give the substituted methoxymethylenemalononitrile (V) .
  • V methoxymethylenemalononitrile
  • VI 3-substituted-5-aminopyra- zole-4-carbonitrile
  • V I 3-substituted-5- 0 minopyrazole-4-carboxamide
  • 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 5 (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 l-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 formula I and/or at least one and up to all of their nucleotides from the substituted nucleotide bases of formula I' .
  • 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 DNA 0and 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,Jf.-diisopropyl)phosphoramidite cyanoethyl ester derivatives, and incorporated into synthetic oligonucleotides following the procedures in "Oligonucleotide Synthesis: A Practical Approach", supra .
  • the _ ⁇ *.-protecting groups are then removed, along with the other oligonucleotide blocking groups, by post-synthesis aminolysis, by procedures generally known in the a t.
  • the activated nucle tides- may ⁇ be used directly on an automated DNA synthesizer according to the procedures and instructions of the particular synthesizer employed.
  • the oligonucleo ⁇ tides may be prepared on the synthesizer using the standard commercial phosphoramidite or H-phosphonate chemistries.
  • the amino- pyrazolopyrimidine nucleotide triphosphates may substitute for an adenine using the nick translation procedure, as described by Langer et al. , Proc. Natl. Acad. Sci. USA. 211:6633-6637 (1981), the disclosure of which is incorporated herein by reference.
  • the leaving group such as a haloacyl group
  • addition of an ⁇ -haloacet- amide may be verified by a changed mobility of the modified compound on HPLC, corresponding to the removal 5 of the positive charge of the amino group, and by sub ⁇ sequent 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: bromoacetyl, iodoacetyl and the less reactive but c ⁇ nfor- mationally more flexible 4-bromobutyryl. Bromoacetyl ' and 15iodoacetyl were found to be of equal reactivity in crosslinking.
  • An oligonucleotide probe according to the invention includes at least one labeled substituted pyrazolo[3,4-d] yrimidine nucleotide moiety of formula I 20and/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- he-use of autoradiography with 3 H, 125 I, 35 S, 25 14 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, 30enzymes 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.
  • Radio ⁇ active probes are typically made using commercially available nucleotides containing the desired radioactive 5 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 5 or enzyme) or labeled indirectly by conjugation with a ligand.
  • a signal e.g., fluorophore, chemiluminescent agent 5 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 covalently bound to a detectable signal, such as an oenzyme or photoreactive compound.
  • Ligands and anti- ligands may be varied widely.
  • a ligand has a natural "antiligand", namely ligands such as biotin, thyroxine, and cortisol, it can be used in conjunction with.,j.ts—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 is Oincorporated 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 include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, rare earths, etc. 3/03736
  • Chemiluminescers include luciferin, acridinium esters and 2,3-dihydrophthalazinediones, e.g., luminol.
  • hybridization conditions are not critical and will vary in accordance with theomme- 5 gator'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 10 chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris HC1, 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 15 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 20 o.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-sacch_-_5_Ldic polymers, such as dextran sulfate.
  • 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, 5 substantial excesses of probe over the stoichiometric amount of the target nucleic acid will be employed to 03736
  • 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 75 ⁇ C. 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 concen ⁇ tration of formamide within the range of about 20% to about 50%.
  • Af-te_f hybridization at a temperature and time period appropriate for the particular hybridization solution used, 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.
  • reagents e.g., sodium chloride, buffers, organic solvents and detergent
  • These reagents may be at similar concentra ⁇ tions as the hybridization medium, but often they are at lower concentrations when more stringent washing condi ⁇ tions are desired.
  • the time period for which the support is maintained in the wash solutions may vary from minutes to several hours or more. 93/03736 r ⁇
  • 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. 5
  • 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 0 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 sample 5 is detected by incubation with an appropriate substrate for the enzyme.
  • the signal generated may be a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemilumin- escence.
  • the preferred label for dipstick assays generates a colored precipitate to indicate a positive reading.
  • alkaline phosphatase will dephos- phorylate indoxyl phosphate which then will participate in a reduction reaction to convert tetrazolium salts to highly, colored—and .insoluble formazans.
  • Detection of a hybridization complex may require the binding of 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-conjugated Probe 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 radio ⁇ active 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 1 .
  • the method comprises the steps of:
  • 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 o 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 5 system, such as an enzyme for example, and a substrate for the system.
  • ODNs Oligodeoxynucleotides
  • mRNAs complementary messenger RNAs
  • antisense ODNs are believed to inhibit the processing or translation of message primarily through an RNase H-mediated cleavage of the target mRNA sequence. Because of this inhibitory effect, ntisense ODNs may be useful as anti-viral, anti- parasitic, and anti-cancer agents. Further, antisense ODNs provide a unique opportunity for rational drug development, since a genetic target offers bothaki 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 deleterious effects on other properties may be difficult.
  • antisense ODNs with modified backbones 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 free 0radical-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 RNA ⁇ inactive-r 5 Cellular uptake of antisense ODNs is another area of concern.
  • While charged ODNs may be actively taken up into mammalian cells through an energy dependent pathway, augmentation of this pathway to achieve high intracellular concentrations of antisense ODN is ⁇ desirable.
  • "Endocytic approaches" which enhance 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 Uposomes; and linkage of ODNs to soluble macromolecular complexes.
  • Application of the above-noted approaches, alone or in combination may provide an increase in antisense ODN potency in the range of 1000-fold and may significantly reduce the cost of ODN synthesis. 5
  • anti-gene A variation of the "antisense” approach to rational drug design is termed "anti-gene”.
  • antisense ODNs target single stranded mRNA
  • anti-gene 0ODNs 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 5selected 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 In contrast, the target for (antisense ODNs, mRNA, represents a very small subset of the information encoded in DNA.
  • anti-gene ODNs have broader applicability and are potentially more powerful than antisense ODNs that merely inhibit mRNA processi-ng-and— ranslation.
  • 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. Based on the known stabilities of the two target nucleic acid species (i.e., DNA and RNA), 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 potentially high copy number of such mRNA species suggest that anti-sense ODNs will provide relatively short term effects. While cellular uptake of anti-gene ODNs may 5 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 o 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 5 triplets below). In each case, the third strand base is presented first and is followed by the base pair; hydrogen bonding between the first two bases maintains the third base interaction.
  • triple-stranded complexes are generally less stable than the parental double-stranded DNA, which is maintained by a combination of two (A-T) or three (G-C) hydrogen bonds between purine and pyrimidine pairs.
  • Cytosine/thy idine-, 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 0reverse 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 anti- 5parallel to the homopurine strand of the duplex, depending on the G content of the third strand. These 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 ohomopurine 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 5Strand to recognize T-A base pairs, this interaction involves only one hydrogen bond and is relatively unstable.
  • anti-gene ODNs may be modified with a variety of pendant groups designed to augment their activity.
  • intercalating groups, cleaving agents, and crosslinking moieties may be appended to the termini of anti-gene ODNs.
  • these groups interact with the adjacent duplex and intercalate, cleave or crosslink, respectively.
  • substitution of 5-methyl cytosine for cytosine in the third strand ODN significantly stabilizes triplexes formed with "G"-rich /03736
  • ODNs with modified backbones such as oligonucleoside methyl-phosphonates and phosphorothioates, may form triple-stranded complexes.
  • a important disadvantage of triple strand formation as discussed above is the relatively slow 0 inetics 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 5provides 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) .
  • Homologous recombination in E. coll serves as an illustrative example of processes that occur in both procaryotic and eucaryotic cells.
  • Studies of purified recombinational 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.
  • "presynapsis” single stranded circular DNA is coated with the multifunctional protein recA in the presence of ATP.
  • the resultant 36 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") .
  • Paranemic joints are highly unstable and upon deproteinization readily dissociate.
  • 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 (removal of recA) .
  • the plectonemically coiled synaptic complex contains 18 triads per right handed helical turn. While recA is a critical part of the complex, sequence specificity resides entirely in the hydrogen bonding between bases.
  • a plectonemically coiled synaptic complex two alternative conformations exist. In one, the negative linear strand of parental DNA is Watson Crick base-paired to the positive linear strand; in the other, the negative linear strand of parental DNA is Watson Crick base-paired to the positive circular single strand. In both conformations, the bases of the third strand are presumed to be hydrogen-bonded in a Hoogstein or reverse Hoogstein fashion to the purines of the other 37 two strands (according to the binding code described above) .
  • the single stranded circular DNA within the triple stranded nucleoprotein filament subsequently displaces the homologous linear strand ("strand 5exchange") .
  • This displacement features a recA-catalyzed, energy-dependent release of the linear positive strand, coupled with hybridization of the circular single strand to the complementary linear strand.
  • recA remains associated with the locomplex.
  • recA dissociates from the newly formed double strand and recombination is complete.
  • the protein-free triple stranded DNA complexes are essentially resistant to single strand-specific nucleases 15and exhibit very high T_'s. This is likely attributable to hydrogen bonding of the so-called "third strand” to both parental strands, as well as to the highly underwound state of the complex.
  • the highly underwound, deproteinized synaptic complexes therefore are analogous 0 to highly underwound recA-containing complexes.
  • recA may catalyze the formation of a DNA triple strand complex not otherwise attainable.
  • the two positive strands have identical sequence and positive strand is capable of forming a double stranded hybrid with the negative parental strand.
  • 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._a,ny)-o-f—the-targeted double-stranded DNA, the resultant synaptic complex is classified as a proximal joint, a medial joint, or a distal joint.
  • 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' to 53' 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 formation (even using a DNA target and ODN with a very small region of shared homology) may be obtained using linear targets. Selection of a synthetic, oligomeric double stranded target DNA versus a supercoiled target in the model system will vary with the characteristics of the target duplex DNA.
  • 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 s-trand-cBNA sequence; upon recognition and binding of 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 of the two duplex strands in the target DNA.
  • Anti-gene ODNs (of the appropriate polarity) may be used in combination with endogenous recombinatory pathways to form sequence-specific triple strand complexes with chromosomal DNA.
  • anti-gene ODNs of a /03736 may be used in combination with endogenous recombinatory pathways to form sequence-specific triple strand complexes with chromosomal DNA.
  • 40 variety of lengths may be complexed with a recombinational enzyme prior to combination with target double strand DNA.
  • 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.
  • the cellular recombinational pathway is 0being harnessed, the DNA in higher order chromatin structures will be accessible for targeting.
  • the resultant triple strand complexes display increased stability with increased anti-gene ODN length.
  • 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) e nzyme-catalyzed triple strand formation to achieve inactivation of a target DNA sequence.
  • the ODN has a crosslinking moiety covalently attached thereto.
  • the crosslinking moiety, the target DNA sequence and the environment and characteristics of the target DNA sequence, inactivation of the parental duplex strands may be permanent.
  • a single administration of one or more anti-gene ODNs may abolish the expression of integrated retroviral genomes, of episomal herpesvirus genomes, or of mutant oncogenes.
  • the modified target DNA no longer supports replication or transcription. Unlike all other lesions in DNA, however, this modification is not repairable.
  • crosslinked DNA is repaired by a combination of excision repair and homologous recombination.
  • crosslinked triple strand complexes there will be no undamaged copies of the targeted gene to 5 participate in recombination.
  • 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 o by the resultant mutations.
  • recombination enzymes in combination- with anti-gene ODNs significantly enhances the efficiency with which the single strand ODN "finds" its complementary target DNA sequence. Accordingly, the 5 efficiency of triple strand formation is greatly increased when the 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 transcription or replication.
  • 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 /03736
  • Preferred recombination enzymes include procaryotic and eucaryotic recombination enzymes, such as 5 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 o 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 5 administer the nucleoprotein filament in a liposome.
  • Thin layer chromatography was performed on silica g-e-l-60-r*F--254 plates (Analtech) using the following 5S ⁇ lvent 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 on 0an Applied Biosystems Model 380B Synthesizer. Oligo ⁇ nucleotides were isotopically labeled using T4 Polynucleotide kinase (BRL) and ⁇ - 32 P-ATP (New England Nuclear) .
  • T4 Polynucleotide kinase BDL
  • ⁇ - 32 P-ATP New England Nuclear
  • 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 IN HC1 and the organic layer evaporated to dryness.
  • the residue was suspended in 2-propanol/lN 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 dichloromethane solution was washed with ice cold 2N HC1 (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. l-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 o 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.
  • N-l and N-2 Two major 5products were isolated and identified as the N-l and N-2 isomers in 57% (3.6 g) and 20% (1.2 g) N-l and N-2 yields, respectively. Approximately 1 g of a mixture of N-l and N-2 isomers was also collected. Overall yield of glycosylated material was 5.8 g (92%).
  • Example 8 The monophosphate of Example 8 (80 mg, ca. 0.1_ mmole) was dissolved in DMF with the addition of 0triethylamine (14 ⁇ L) . Carbonyldiimidazole (81 mg, 0.5 mmole) was added and the. solution stirred at RT for 18 hr. The solution was treated with methanol (40 ⁇ L) , and after stirring for 30 min tributylammonium pyrophosphate (0- « . 5_S in_-0.__5_.mL-DMF) was added. After stirring for 24 5hr another aliquot of tributylammonium pyrophosphate was added and the solution was stirred overnight.
  • 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
  • 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.
  • EXAMPLE 11 5-Amino-3-[(5-trityla_ ⁇ ino)pentyl]pyrazole-4-carboxamide.
  • Example 5 Following the procedure of Example 5, the pyrazolopyrimidine of Example 12 is treated with sodium hydride and reacted with l-chloro-l,2-dideoxy-3,5-di-0- toluoylribofuranose. The resulting compound is reacted with MCPBA and with ethanolic ammonia, and the toluoyl oprotecting groups are removed to give the product.
  • EXAMPLE 14 l-(2-Deoxy-/--D-eryt_.ropentofuranosyl)-4-hydroxy-3-[5-(6- biotinamido) exanamidopentyl]pyrazolo[3,4-d] yrimidin-6- 5aX52ne 5'-monophosphate.
  • Example 13 Following the procedure of Example 1 , the pyrazolopyrimidine of Example 13 is reacted with phosphoryl chloride to give the corresponding 5'-mono- 0phosphate.
  • EXAMPLE 15 0 1-(2-Deoxy-?-D-erythropentofuranosyl)-4-hydroxy-3-[5-(6- biotinamido)hexanamidopentyl]pyrazolo[3 ,4-cl]pyrimi in-6- amine 5'-triphosphate.
  • Example 16 the benzoylamine of Example 16 is treated with palladium hydroxide on carbon and then with trifluoroacetic anhydrid____to_-g___*/e l-(2-deoxy-?-D-erythropentofuranosyl)- 3-[5-(trifluoroacetamido)pentyl]pyrazolo[3,4-d]pyrimi- dine-4-benzoylamine.
  • EXAMPLE 18 l-(2-Deoxy-5- ⁇ -dimethoxytrityl-/9-D-erythropentofurano- syl)-3-[5-(trifluoroacetamido) entyl]pyrazolo[3,4-d]- pyrimidine-4-benzoylamine 3'- ⁇ -(N,N-diisopro ⁇ yl)phos ⁇ phoramidite cyanoethyl ester.
  • Example 17 The compound of Example 17 is reacted with dimethoxytrityl chloride and pyridine to give the corresponding 5'-dimethoxytrityl compound. This compound is then reacted with cyanoethyl chloro-N,N-diisopropyl- 3/03736
  • reaction mixture is evaporated to dryness and purified by chromatography to give 5-(3-iodoacetamidopropyl)-2'- deoxyuridine.
  • Nucleosides were 5'-dimethoxytritylated, following known procedures, to give around 85% yield, and the 3 *-phosphoramidite was made using diisopropylamino /?- cyanoethylchlorophosphite (as described in "Oligonucleotide Synthesis: A Practical Approach", supra) with di sopr.op._u.ethylamine in methylene chloride.
  • the phosphoramidite was made into a 0.2N solution in acetonitrile and placed on the automated DNA synthesizer. Incorporation of these new and modified phosphoramidites gave incorporation similar to ordinary phosphoramidites (97-99% as judged by assay of the trityl color released 0 by UV.)
  • 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 during --concentration. The oligonucleotide was evaporated to dryness under vacuum and redissolved in 1.0 mL water. The oligonucleotides were purified by HPLC using 15-55% /03736
  • nucleoside 5-(3-trifluoroacetamidoprop-1-yl)-2'-deoxyuridine was converted to the 5'-O-dimethoxytrityl-3 '-(N,N- diisopropyl)phosphoramidite cyanoethyl ester derivative.
  • a corresponding 14-mer oligonucleotide was also prepared where U 1 is the unmodified deoxyuridine.
  • n- hydroxysuccinimide haloacylate such as ⁇ -haloacetate or 03736
  • haloacyl moiety was examined by HPLC.
  • a Zorbax® oligonucleotide column (Dupont) eluted with a 20 minute gradient of 60% to 80% B composed of: A (20% acetonitrile:80% 0.02 N NaH 2 PO and B (1.2 N NaCl in 20% acetonitrile:80% 0.02 N NaH 2 PO .
  • the presence of a reactive ⁇ -haloacyl moiety was indi ⁇ cated by return of the retention time of the ⁇ -haloacyl- 5amidoalkyl oligonucleotide to the corresponding amino- alkyl oligonucleotide after exposure to IN cysteamine.
  • 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 iodoacet- amidopropyl 14-mer (oligo B) , 10.09 min.
  • the a inobutyl 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-l-yl)-2•- c deoxyuridine or 5-(4-(4-bromobutyramido)but-l-yl)-2'- deoxyuridine, respectively.
  • the reaction of crosslinking a DNA probe to a target nucleic acid sequence contained 1 ⁇ g of haloacyl- camidoalkyl probe and 10 ng of 32 P-labeled cordycepin- tailed 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. These 0solutions 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-l-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. Sci. USA. 2__. : -560 (1977), this treatment quantitatively cleaves the target strand 3'- to the site of alkylation.
  • Psoralen-modified, photo-crosslinkable ODNs 5 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 1Q unique 5'-TpA-3' sequence. Following partial photoreversal of the crosslink by brief exposure to 260 nm light, the HMT furan-side onoadducted ODN was isolated by denaturing PAGE.
  • HMT 4 '-hydroxymethy-4,5',8-trimethylpsoralen
  • a psoralen 1 .. 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 linker arm that spans the 5' terminus of the ODN and the 4 • position of the 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.
  • TMP is modified at its 4 ' position with linker which is attached to the C8 position of deoxyadenosine.
  • 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- o 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 introduce a sequence-specific crosslink the complementary site in a double stranded DNA target.
  • the ODN which 5 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 By linking the psoralen through the 5' position, the furocourmain is able to readily intercalate into a double stranded 5'-TpA-3' 0 sequence immediately adjacent to the triple strand. Both the furan and pyrone rings are properly positioned to photoreact with thymidines.
  • the length of the psoralenated tail may be altered to obtain optimal -• inter.calation-"-c ⁇ f-the psoralen into the duplex-triple 5 strand junction.

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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.
EP92918930A 1991-08-21 1992-08-19 Oligonucleotides a reticulation pour la formation de triple brin a mediation enzymatique. Withdrawn EP0661979A4 (fr)

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US5824796A (en) * 1988-09-28 1998-10-20 Epoch Pharmaceuticals, Inc. Cross-linking oligonucleotides
USRE38416E1 (en) 1988-09-28 2004-02-03 Epoch Biosciences, Inc. Cross-linking oligonucleotides
US5849482A (en) * 1988-09-28 1998-12-15 Epoch Pharmaceuticals, Inc. Crosslinking 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
IL104965A (en) * 1992-03-05 1999-11-30 Isis Pharmaceuticals Inc Coupled oligonucleotides cross-linked
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
EP0695306A1 (fr) * 1993-04-19 1996-02-07 Gilead Sciences, Inc. Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
US5801155A (en) 1995-04-03 1998-09-01 Epoch Pharmaceuticals, Inc. Covalently linked oligonucleotide minor grove binder conjugates
US6312894B1 (en) 1995-04-03 2001-11-06 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
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
US5948653A (en) * 1997-03-21 1999-09-07 Pati; Sushma Sequence alterations using homologous recombination
US5770716A (en) * 1997-04-10 1998-06-23 The Perkin-Elmer Corporation Substituted propargylethoxyamido nucleosides, oligonucleotides and methods for using same
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
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
CA2542768A1 (fr) 2003-10-28 2005-05-12 Epoch Biosciences, Inc. Sondes fluorescentes pour la detection d'adn par hybridation a sensibilite amelioree et bruit de fond faible

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