Classifications

• • 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
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
• • 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
• • 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
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/708—Specific hybridization probes for papilloma

Definitions

• This invention relates to nucleoside crosslinking agents and to the use of these compounds in the preparation of oligonucleotides.
• 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.
• some 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.
• a variety of recent studies using plasmid vectors for the introduction of antisense RNAs into eukaryotic cells have shown that they effectively inhibit expression of mRNA targets in vivo (reviewed in Green, et al., Ann. Rev. Bioche . 55: 569-597 (1986)).
• 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 (Paterson, et al., Proc. Natl. Acad. Sci 74: 4370-4374
• Zamecnik and Stephenson Proc. Natl. Acad. Sci. USA. 25:280 (1978)
• Zamecnik and Stephenson Proc. Natl. Acad. Sci. USA. 25:280 (1978)
• a small antisense oligodeoxynucleotide probe can inhibit replication of Rous Sarcoma Virus in cell culture, and that RSV viral RNA translation is inhibited under these conditions.
• 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 oligonucieotide 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 oligonucieotide analog, an 8-nucleotide sequence complementary to the acceptor splice junction of a mRNA of Herpes simplex virus, Type 1 can inhibit virus replication in intact Vero cells. However, fairly high concentrations (>25 mM) of this nonionic probe were required for this inhibition.
• crosslinking oligonucleotides in the chemotherapeutic field might be of great significance, their impact in DNA probe-based diagnostics is of equally great importance.
• the ability to covalently crosslink probe-target hybrids has the potential to dramatically improve background and sensitivity limits in diagnostic assays as well as permit novel assay formats.
• crosslinking suggests potential problems that must be circumvented.
• the oligonucieotide 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.
• oligonucieotide that, upon hybridization, results in a duplex whose T m is just above the physiological temperature of 37'C.
• T m is just above the physiological temperature of 37'C.
• the optimization can be accomplished by judicious choice of oligonucieotide 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 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 crosslinking nucleic acid hybridization probes.
• W08503075 describes crosslinking disulfonic esters useful as nucleic acid fragmentation agents.
• DE3310337 describes the covalent crosslinking of single-stranded polynucleotides 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.
• 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 hybridization assays.
• the oligonucleotides may also be used 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 specificity.
• 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.
• crosslinking agents of this invention are derivatives of nucleotide bases with a crosslinking arm and are of the following formula (I'):
• R-L is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached via oxygen to the sugar moiety by an oxygen and including groups 0-, Q- and Q ⁇ , 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 1 , O-R', or N-R'R"; each of R 1 and R" is independently hydrogen or C ⁇ gal yl; B is a nucleic acid base or analog thereof that is a component of an oligonucieotide;
• 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 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.
• the covalent crosslinking arm of the 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 oligonucieotide probe comprising at least one of a labeled nucleotide base of the present invention.
• 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 P labeled HPV target and crosslinked product following cleavage at the 3 ' side of the crosslinked guanosme.
• Lane 1 32P-labeled
• Figure 3 depicts an autoradiogram of 32P labeled HPV target and crosslinked product showing hybrid separation by denaturing polyacrylamide gel electrophoresis.
• Lane l
• Control 32P-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 (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 via oxygen to the sugar moiety by an oxygen and including groups Q ⁇ , Q 2 and Q_, or with a reactive precursor thereof suitable for nucleotide bond formation;
• Q 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-L_ 6 alkyl;
• B is a nucleic acid base or analog thereof that is a component of an oligonucieotide
• 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-'0-methylribose and embraces either ano er, or ⁇ .
• the phosphorus derivative attached via oxygen 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 oligonucieotide.
• 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 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, and the deazapurines. 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]pyrimidin-5-yl, where the purines are attached to the sugar moiety of the oligonucleotides via the 9-position, the pyrimidines via the 1-position, and the pyrrolopyrimidines via the 7-position.
• such functionalities including aliphatic or aromatic amines, exhibit nucleophilic properties and are capable of serving as a point of attachment of the -(CH 2 ) -A' group.
• Amino groups and blocked derivatives thereof are preferred.
• the leaving group A' may be chosen from, for example, such groups as chloro, bro o, iodo, S0 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-__ 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.
• 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 and reacting with the N-7 of 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 oligonucieotide containing UZC (preferably UCC) , where TJ is dUrd 5-substituted with the crosslinking arm.
• the adenme-modified AZ C triplet would target GZ T, where Z is any base. It has been found that when the modified base containing the crosslinking arm is a uracil and the target sequence is GGA, alkylation of the second guanine on the target's 5' side of the crosslinker-modified base pair is the exclusive action observed (as shown in Figure 1) . The crosslinking reaction seems to be very specific for the
• the 5-(substituted)-2'-deoxyuridines may be prepared by the routes shown in Schemes 1 and 2.
• the moiety Y' in Schemes 1 and 2 refers to -(Y) r -(CH 2 ) m -A' .
• 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, a or ⁇ .
• the phosphorus derivative attached via oxygen 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 oligonucieotide.
• 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 alkylphosphoramidites. Alternatively, activated phosphate diesters may be employed for this purpose.
• 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 oligonucieotide to the target sequence. This can be accomplished by crosslinking agents being covalently attached to the oligonucieotide, 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 of the invention When oligonucleotides 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 oligonucieotide 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.
• Oligonucleotides of the present invention may comprise at least one and up to all of their nucleotides from the substituted nucleotide bases of formula I• .
• protective groups are introduced onto the nucleosides of 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,
• the activated nucleotides are incorporated into oligonucleotides in a manner analogous to that for DNA and 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 enzy atically or via chemical synthesis.
• the nucleotides may be converted to their 5•-O-dimethoxytrityl-3 ' - (N,N- diisopropyl)phosphoramidite cyanoethyl ester derivatives, and incorporated into synthetic oligonucleotides following the procedures in "Oligonucieotide Synthesis: A Practical Approach", supra .
• the W-protecting groups are then removed, along with the other oligonucieotide blocking groups, by post-synthesis aminolysis, by procedures generally known in the art.
• the activated nucleotides 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.
• 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-amino- ethanethiol to give a derivative with reverse phase HPLC mobility similar to the original aminoalkyloligonucleotide.
• each of the following electrophilic leaving groups were attached to an aminopropyl group on human papillomavirus (HPV) probes: bromoacetyl, iodoacetyl and the less reactive but conformationally more flexible 4-bromobutyryl. Bromoacetyl and iodoacetyl were found to be of equal reactivity in crosslinking. Oligonucieotide Probe Labelling
• An oligonucieotide probe according to the invention includes 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, 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, 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 depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.
• 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 poiymerase in the presence of radioactive dNTP's, or by transcribing RNA from templates using RNA poiymerase 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 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 enzyme or photoreactive compound.
• Ligands and antiligands 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 is incorporated 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.
• Chemiluminescers include luciferin, acridiniu esters and 2,3-dihydrophthalazinediones, e.g., luminol.
• Hybridization and Crosslinking of Probe and Target - Assays The specific hybridization and crosslinking conditions are not critical and will vary in accordance with the investigator's preferences and needs.
• Various hybridi ⁇ zation 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 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 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 anioni ⁇ polyacrylate or polymethylacrylate, and charged saccharidic polymers, such as dextran sulfate.
• the particular 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.. €>3_:378-383 (1969); and John et al.. Nature. 22J3:582-587 (1969) . As improvements are made in hybridization techniques, they can readily be applied.
• 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 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 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 support is 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.
• the preferred label for dipstick assays generates a colored precipitate to indicate a positive reading.
• alkaline phosphatase will dephosphorylate 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. Typically, such 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 generated 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 oligonucieotide probe including at least one labeled substituted nucleotide moiety of formula I'.
• the method comprises the steps of:
• oligonucieotide probe including at least one labeled substituted nucleotide moiety of formula I• , wherein the probe comprises a sequence complementary to that of the target nucleic acids, under conditions which permit crosslinking of probe and target;
• the above method may be conducted following procedures well known in the art.
• kits for carrying out the invention.
• an assay may be provided in kit form.
• a typical kit will include a probe reagent component comprising an oligonucieotide including at least one labeled nucleotide moiety of formula I', the oligonucieotide 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.
• EXAMPLE 1 5-(4-Phthalimidobut-l-yn-l-yl)-2'-deoxyuridine.
• Nucleosides were 5'-dimethoxytritylated, following known procedures, to give around 85% yield, and the 3•-phosphoramidite was made using diisopropylamino /3-cyanoethylchlorophosphite (as described in "Oligonucieotide Synthesis: A Practical Approach", supra) with diisopropylethylamine in ethylene 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
• 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 oligonucieotide was evaporated to dryness under vacuum and redissolved in 1.0 L water. The 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.
• oligonucieotide was collected and evaporated to dryness, then it was redissolved in 80% aqueous acetic acid for 90 minutes to remove the trityl group. Desalting was accomplished with a G25 Sephadex column and appropriate fractions were taken. The fractions were concentrated, brought to a specific volume, dilution reading taken to ascertain overall yield and an analytical HPLC done to assure purity. Oligonucleotides were frozen at -20 ⁇ C until use. Following the above procedures, the nucleoside
• a corresponding 14-mer oligonucieotide was also prepared where U is the unmodified deoxyuridine.
• n-hydroxysuccinimide haloacylate such as ⁇ -haloacetate or 4-halobutyrate
• 10 ⁇ L of 0.1 M borate buffer, pH 8.5 was incubated at ambient temperature for 30 min. in the dark.
• the entire reaction was passed over a NAP-10 column equilibrated with and eluted with distilled water. Appropriate fractions based on UV absorbance were combined and the concentration was determined spectrophotometrically.
• aminobutyl 14-mer (oligo C, Example 5) was reacted with either N-hydroxysuccinimide ⁇ -iodoacetate or
• the reaction of crosslinking a DNA probe to a target nucleic acid sequence contained 1 ⁇ g of haloacylamidoalkyl probe and 10 ng of 32P-labeled cordycepm-tailed target in
• 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. Results of the reaction of HPV target with a limiting amount of crosslinking probe containing a 5-(3-iodoacetamidopropyl) sidearm are shown in Figure 2. Analysis of the cleavage pattern on a denaturing PAGE gel showed the loss of the crosslinked hybrid with the concomitant appearance of a discrete low molecular weight band.
• Example 7 the crosslinked HPV hybrid of Example 7 (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 f 24:560 (1977), this treatment quantitatively cleaves the target strand 3'- to the site of alkylation.
• the resulting data indicated that the alkylation of the second guanine above the crosslinker-modified base pair (i.e., the guanine above the target base) was the exclusive action observed, indicating that the crosslinking reaction in the HPV model system is remarkably specific.

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