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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
  • A61K47/557—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
• • 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
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

• Gene expression in diseased and healthy individuals is oftentimes different and characterizable.
• the ability to monitor gene expression in such cases provides medical professionals with a powerful diagnostic tool. This form of diagnosis is especially important in the area of oncology, where it is thought that the overexpression of an oncogene, or the underexpression of a tumor suppressor gene, results in tumorogenesis. See Mikkelson et al. J Cell. Biochem. 1991, 46, 3-8.
• nucleic acid e.g., mRNA
• the nucleic acid is chemically or biochemically labeled with a detectable moiety and allowed to hybridize with a localized nucleic acid of known sequence sometimes, know here as a probe.
• the detection of a labeled nucleic acid at the probe position indicates that the targeted gene has been expressed. See, e.g., International Application Publication Nos.WO 97/27317, WO 92/10588 and WO 97/10365.
• the labeling of a nucleic acid is typically performed by covalently attaching a detectable group (label) to either an internal or terminal position.
• Petrie et al. disclosed a dATP analogue, 3-[5-[(N-biotinyl-6- aminocaproyl)-amino]pentyl] - 1 -(2-deoxy- ⁇ -D-erythro-pentofuranosyl)- 1 H- pyrazolo[3,4-d]pyrimidin-4-amine-5'-triphosphate. Bioconjugate Chern. 1991, 2, 441-446. The analogue, shown below, is modified at the 3-position with a linker arm that is attached to a biotin moiety. Petrie et al. reported that the compound wherein R is biotin is incorporated into DNA by nick translation.
• Prober et al. disclosed a set of four dideoxynucleotides, each containing a succinylfluorescein dye. Science 1987, 238, 336-341.
• the dideoxynucleotides one of which is shown below, were enzymatically incorporated into an oligonucleotide through a template directed extension of a primer.
• the compounds provided for a DNA sequencing method based on gel migration.
• Herrlein et al. disclosed modified nucleoside trisphosphates of the four DNA bases. Helv. Chim. Acta 1994, 77, 586-596. The compounds, one of which is shown below, contain a 3'-amino group containing radioactive or fluorescent moieties. Herrlein et al. further described the use of the nucleoside analogues as DNA chain terminators.
• Cech et al. disclosed 3 '-amino-functionalized nucleoside triphosphates.
• nucleic acid labeling compounds that are effectively incorporated into a nucleic acid to provide a readily detectable composition would benefit genetic analysis technologies. It would aid, for example, in the monitoring of gene expression and the detection and screening of mutations and polymorphisms.
• a compound should be suitable for incorporation into a nucleic acid either by enzymatic or other means.
• the nucleic acid to which the labeling compound is attached should maintain its ability to bind to a probe, such as a complementary nucleic acid.
• nucleic acid labeling compounds for use as coupling agents for probes are available there is a continuing need for additional compounds that are more efficient labeling compounds. There also exists a need for compounds that have increased solubility. This will make the compounds more useful for monitoring gene expression.
• the present invention relates to nucleic acid labeling compounds. More specifically, the invention provides heterocyclic derivatives containing a detectable moiety. The invention also provides methods of making such heterocyclic derivatives. It further provides methods of attaching the heterocyclic derivatives to a nucleic acid.
• the nucleic acid labeling compounds of the invention the general formula
• X is O, S, NRi or CHR , wherein Rr and R 2 are, independently, H, alkyl or acid by either enzymatic or, e.g., by chemical means;
• Y is H, N 3 , F, OR 9 , SR 9 or NHR 9 , wherein R 9 is H, alkyl or aryl;
• Z is H, N 3 , F or OR10, wherein Rio is H, alkyl or aryl;
• the nucleic acid labeling compounds of the present invention are heterocyclic derivatives that have a detectable moiety.
• the invention also provides methods of making such heterocyclic derivatives. It further provides methods of attaching the heterocyclic derivatives to a nucleic acid.
• the present invention provides nucleic acid labeling compounds that are capable of being enzymatically incorporated into a nucleic acid.
• the nucleic acids to which the compounds are attached substantially maintain their ability to bind to a complementary nucleic acid sequence.
• Figure 1 shows a nonlimiting set of template moieties.
• Figure 2 shows a nonlimiting set of heterocyclic groups: 4- aminopyrazolo[3,4-d]pyrimidine, pyrazolo[3,4-d]pyrimidine, 1 ,3-diazole (imidazole), l,2,4-triazine-3-one, l,2,4-triazine-3,5-dione and 5-amino- 1,2,4- triazine-3-one.
• Figure 3 shows a schematic for the preparation of Nl -labeled 5-( ⁇ -D- ribofuranosyl)-2,4[lH,3H]-pyrimidinedione 5'-triphosphate.
• Figure 4 shows HIV array data for analog 42a (T7 labeling of RNA target).
• Figure 5 shows HPLC incorporation efficiency of C-nucleotide 42a (T7 RNA pol, 1 kb transcript).
• Figure 6 shows INT incorporation of saturated versus unsaturated nucleic acid labeling compounds.
• a general aspect of the instantly disclosed invention includes nucleic acid labeling compounds of formula (II):
• the connecting group may optionally not be present, depending on, inter alia, the nature of L and Q.
• nucleic acid labeling compounds have formula (HI):
• A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid by either enzymatic or, e.g., by chemical means;
• X is O, S, NRi or CHR 2 , wherein Ri and R 2 are, independently, H, alkyl or aryl;
• Y is H, N 3 , F, OR 9 , SR 9 or NHR 9 , wherein R 9 is H, alkyl or aryl;
• Z is H, N 3 , F or ORio, wherein Rio is H, alkyl or aryl; L is linker group;
• nucleic acid labeling compounds have formula (IV):
• A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid by either enzymatic or, e.g., by chemical means;
• X is O, S, NRi or CHR 2 , wherein Ri and R are, independently,
• Y is H, N 3 , F, OR 9 , SR or NHR 9 , wherein R 9 is H, alkyl or aryl;
• Z is H, N 3 , F or ORio, wherein Rio is H, alkyl or aryl; L is functionalized alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
• the nucleic acid labeling compounds used in the coupling have the following structures for example: or
• A is H or a functiona group t at perm ts t e attachment of the nucleic acid labeling compound to a nucleic acid;
• X is O, S, NRi or CHR 2 , wherein Ri and R 2 are, independently, H, alkyl or aryl;
• Y is H, N 3 , F, OR 9 , SR 9 or NHR 9 , wherein R 9 is H, alkyl or aryl;
• Z is H, N 3 , F or ORio, wherein Rio is H, alkyl or aryl; L is linker group;
• Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
• the hybridization product formed from this nucleic acid derivative comprises the nucleic acid derivative bound to a complementary probe.
• the probe is attached to a glass chip.
• the nucleic acid labeling compounds used in the coupling have the following structures for example:
• A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid
• X is O, S, NRi or CHR 2 , wherein Ri and R 2 are, independently, H, alkyl or aryl
• Y is H, N 3 , F, OR 9 , SR 9 or NHR 9 , wherein R 9 is H, alkyl or aryl
• Z is H, N 3 , F or ORio, wherein R1 0 is H, alkyl or aryl
• L is functionalized alkyl
• Q is a detectable moiety
• M is a connecting group, wherein m is an integer ranging from 0 to about 3.
• the hybridization products formed from the nucleic acid derivatives of the invention comprise the nucleic acid derivative bound to a complementary probe.
• the probe is attached to a glass chip.
• the method of nucleic acid detection using the nucleic acid derivatives of the inventioin involves the incubation of the derivative with a probe.
• the probe is attached to a glass chip.
• the methods of the invention include the steps of: (a) providing at least one nucleic acid coupled to a support; (b) providing a labeled moiety capable of being coupled with a terminal transferase to said nucleic acid; (c) providing said terminal transferase; and (d) coupling said labeled moiety to the nucleic acid using said terminal transferase.
• the methods of the invention include the steps of: (a) providing at least two nucleic acids coupled to a support; (b) increasing the number of monomer units of said nucleic acids to form a common nucleic acid tail on said at least two nucleic acids; (c) providing a labeled moiety capable of recognizing said common nucleic acid tails; and (d) contacting said common nucleic acid tails and said labeled moiety.
• the methods of the invention include the steps of: (a) providing at least one nucleic acid coupled to a support; (b) providing a labeled moiety capable of being coupled with a ligase to said nucleic acid; (c) providing said ligase; and (d) coupling said labeled moiety to said nucleic acid using said ligase.
• This invention also provides compounds of the formulas described herein.
• Alkyl refers to a straight chain, branched or cyclic chemical group containing only carbon and hydrogen. Alkyl groups include, without limitation, ethyl, propyl, butyl, pentyl, cyclopentyl and 2-methylbutyl. Alkyl groups are unsubstituted or substituted with 1 or more substituents (e.g., halogen, alkoxy, amino).
• Aryl refers to a monovalent, unsaturated aromatic carbocyclic group.
• Aryl groups include, without limitation, phenyl, naphthyl, anthryl and biphenyl.
• Aryl groups are unsubstituted or substituted with 1 or more substituents (e.g. halogen, alkoxy, amino).
• amido alkyl refers to a chemical group having the structure -C(O)NR R -, wherein R 3 is hydrogen, alkyl or aryl, and R 4 is alkyl or aryl.
• the amido alkyl group is of the structure -C(O)NH(CH 2 ) n R 5 -, wherein n is an integer ranging from about 2 to about 10, and R 5 is O, NR ⁇ , or C(O), and wherein R 6 is hydrogen, alkyl or aryl. More preferably, the amido alkyl group is of the structure -C(O)NH(CH 2 ) n N(H)-, wherein n is an integer ranging from about 2 to about 6. Most preferably, the amido alkyl group is of the structure -C(O)NH(CH 2 ) 4 N(H)-.
• Alkynyl alkyl refers to a chemical group having the structure -C ⁇ C-R -, wherein R 4 is alkyl or aryl.
• the alkynyl alkyl group is of the structure -C ⁇ C-(CH ) n R 5 -, wherein n is an integer ranging from 1 to about 10, and R 5 is O, NR 6 or C(O), wherein R 6 is hydrogen, alkyl or aryl.
• the alkynyl alkyl group is of the structure -C ⁇ C-(CH 2 ) n N(H)-, wherein n is an integer ranging from 1 to about 4.
• “Functionalized alkyl” refers to a chemical group of the structure -(CH 2 ) n R 7 -, wherein n is an integer ranging from 1 to about 10, and R 7 is O, S, NH or C(O).
• the functionalized alkyl group is of the structure -(CH 2 ) n C(O)-, wherein n is an integer ranging from 1 to about 4. More preferably, the functionalized alkyl group is of the structure -CH 2 C(O)-.
• Alkoxy refers to a chemical group of the structure -O(CH ) n R 8 -, wherein n is an integer ranging from 2 to about 10, and R 8 is O, S, NH or C(O).
• the alkoxy group is of the structure -O(CH 2 ) n C(O)-, wherein n is an integer ranging from 2 to about 4. More preferably, the alkoxy group is of the structure -OCH 2 CH 2 C(O)-.
• Thio refers to a chemical group of the structure -S(CH 2 ) n R 8 -, wherein n is an integer ranging from 2 to about 10, and R 8 is O, S, NH or C(O).
• the thio group is of the structure -S(CH 2 ) n C(O)-, wherein n is an integer ranging from 2 to about 4. More preferably, the thio group is of the structure -SCH 2 CH 2 C(O)-.
• amino alkyl refers to a chemical group having an amino group attached to an alkyl group.
• an amino alkyl is of the structure -NH(CH ) n NH-, wherein n is an integer ranging from about 2 to about 10. More preferably it is of the structure -NH(CH 2 ) n NH-, wherein n is an integer ranging from about 2 to about 4. Most preferably, the amino alkyl group is of the structure -NH(CH 2 ) 4 NH-.
• Nucleic acid refers to a polymer comprising 2 or more nucleotides and includes single-, double- and triple stranded polymers.
• Nucleotide refers to both naturally occurring and non-naturally occurring compounds and comprises a heterocyclic base, a sugar, and a linking group, preferably a phosphate ester.
• structural groups may be added to the ribosyl or deoxyribosyl unit of the nucleotide, such as a methyl or allyl group at the 2'-O position or a fluoro group that substitutes for the 2' -O group.
• nucleic acid may be substituted or modified, for example with methyl phosphonates or O-methyl phosphates.
• Bases and sugars can also be modified, as is known in the art.
• Nucleic acid for the purposes of this disclosure, also includes “peptide nucleic acids” in which native or modified nucleic acid bases are attached to a polyamide backbone.
• Coupled to a support means bound directly or indirectly thereto including attachment by covalent binding, hydrogen bonding, ionic interaction, hydrophobic interaction, or otherwise.
• Probe refers to a nucleic acid that can be used to detect, by hybridization, a target nucleic acid.
• the probe is complementary to the target nucleic acid along the entire length of the probe, but hybridization can occur in the presence of one or more base mismatches between probe and target.
• Perfect match probe refers to a probe that has a sequence that is perfectly complementary to a particular target sequence.
• the test probe is typically perfectly complementary to a portion (subsequence) of the target sequence.
• the perfect match (PM) probe can be a "test probe”, a "normalization control” probe, an expression level control probe and the like.
• a perfect match control or perfect match probe is, however, distinguished from a “mismatch control” or “mismatch probe.”
• perfect match probes are typically preselected (designed) to be complementary to particular sequences or subsequences of target nucleic acids (e.g., particular genes).
• target nucleic acids e.g., particular genes
• the particular target sequences are typically unknown.
• perfect match probe in this context is to distinguish that probe from a corresponding "mismatch control" that differs from the perfect match in one or more particular preselected nucleotides as described below.
• mismatch control or “mismatch probe”, in expression monitoring arrays, refers to probes whose sequence is deliberately selected not to be perfectly complementary to a particular target sequence. For each mismatch (MM) control in a high-density array there preferably exists a corresponding perfect match (PM) probe that is perfectly complementary to the same particular target sequence.
• PM perfect match
• probes are preferably provided as pairs where each pair of probes differ in one or more preselected nucleotides.
• the perfect match and mismatch probes need not be provided as pairs, but may be provided as larger collections (e.g., 3. 4, 5, or more) of probes that differ from each other in particular preselected nucleotides. While the mismatch(s) may be located anywhere in the mismatch probe, terminal mismatches are less desirable as a terminal mismatch is less likely to prevent hybridization of the target sequence.
• the mismatch is located at or near the center of the probe such that the mismatch is most likely to destabilize the duplex with the target sequence under the test hybridization conditions.
• perfect matches differ from mismatch controls in a single centrally-located nucleotide.
• Labeled moiety refers to a moiety capable of being detected by the various methods discussed herein or known in the art.
• the group A is either hydrogen or a functional group that permits the attachment of a nucleic acid labeling compound to a nucleic acid.
• Nonlimiting examples of such groups include the following: monophosphate; diphosphate; triphosphate (H O 9 P); phosphora idite ((R 2 N)(R'O)P), wherein R is linear, branched or cyclic alkyl, and R' is a protecting group such as 2-cyanoethyl; and H-phosphonate (HP(O)O-HNR 3 ), wherein R is linear, branched or cyclic alkyl.
• the heterocyclic group (H c ) is a cyclic moiety containing both carbon and a heteroatom.
• Nonlimiting examples of heterocyclic groups contemplated by the present invention are shown in FIG. 2.: 4-aminopyrazolo[3,4-d]pyrimidine; pyrazolo[3,4-d]pyrimidine; 1,3-diazole (imidazole); l,2,4-triazine-3-one; 1,2,4- triazine-3,5-dione; and, 5-amino-l,2,4-triazine-3-one.
• the linker moiety (L) of the nucleic acid labeling compound is covalently bound to the heterocycle (H c ) at one terminal position. It is attached to the detectable moiety (Q) at another terminal position, either directly or through a connecting group (M).
• linker moieties include amido alkyl groups, alkynyl alkyl groups, alkenyl alkyl groups, - functionalized alkyl groups, alkoxyl groups, thio groups and amino alkyl groups.
• the detectable moiety (Q) is a chemical group that provides an signal.
• the signal is detectable by any suitable means, including spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In certain cases, the signal is detectable by 2 or more means.
• the detectable moiety provides the signal either directly or indirectly.
• a direct signal is produced where the labeling group spontaneously emits a signal, or generates a signal upon the introduction of a suitable stimulus.
• Radiolabels such as H, I, S, C or P
• magnetic particles such as DynabeadsTM, are nonlimiting examples of groups that directly and spontaneously provide a signal.
• Labeling groups that directly provide a signal in the presence of a stimulus include the following nonlimiting examples: colloidal gold (40 - 80 nm diameter), which scatters green light with high efficiency; fluorescent labels, such as fluorescein, texas red, rhodamine, and green fluorescent protein (Molecular Probes, Eugene, Oregon), which absorb and subsequently emit light; chemiluminescent or bioluminescent labels, such as luminol, lophine, acridine salts and luciferins, which are electronically excited as the result of a chemical or biological reaction and subsequently emit light; spin labels, such as vanadium, copper, iron, manganese and nitroxide free radicals, which are detected by electron spin resonance (ESR) spectroscopy; dyes, such as quinoline dyes, triarylmethane dyes and acridine dyes, which absorb specific wavelengths of light; and colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
• a detectable moiety provides an indirect signal where it interacts with a second compound that spontaneously emits a signal, or generates a signal upon the introduction of a suitable stimulus.
• Biotin for example, produces a signal by forming a conjugate with streptavidin, which is then detected. See Hybridization With Nucleic Acid Probes. In Laboratory Techniques in Biochemistry and Molecular Biology; Tijssen, P., Ed.; Elsevier: New York, 1993; Vol. 24.
• An enzyme such as horseradish peroxidase or alkaline phosphatase, that is attached to an antibody in a label-antibody-antibody as in an ELIS A assay, also produces an indirect signal.
• a preferred detectable moiety is a fluorescent group.
• Flourescent groups typically produce a high signal to noise ratio, thereby providing increased resolution and sensitivity in a detection procedure.
• the fluorescent group absorbs light with a wavelength above about 300 nm, more preferably above about 350 nm, and most preferably above about 400 nm.
• the wavelength of the light emitted by the fluorescent group is preferably above about 310 nm, more preferably above about 360 nm, and most preferably above about 410 nm.
• the fluorescent detectable moiety is selected from a variety of structural classes, including the following nonlimiting examples: 1- and 2- aminonaphthalene, p,p'diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p-p'-diaminobenzophenone imines, anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin, perylene, bisbenzoxazole, bis- p-oxazolyl benzene, 1,2-benzophenazin, retinol, bis-3-aminopridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidazolyl phenylamine, 2-oxo-3- chromen, indole, xanthen, 7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins
• fluorescent compounds are suitable for incorporation into the present invention.
• Nonlimiting examples of such compounds include the following: dansyl chloride; fluoresceins, such as 3,6-dihydroxy-9- phenylxanthhydrol; rhodamineisothiocyanate; N-phenyl-l-amino-8- sulfonatonaphthalene; N-phenyl-2-amino-6-sulfonatonaphthanlene; 4-acetamido- 4-isothiocyanatostilbene-2,2'-disulfonic acid; pyrene-3-sulfonic acid; 2- toluidinonapththalene-6-sulfonate; N-phenyl, N-methyl 2-aminonaphthalene-6- sulfonate; ethidium bromide; stebrine; auromine-0,2-(9'-anthroyl)palmitate; dansyl phosphatidylethanolamin; N,N'
• the fluorescent detectable moiety is a fluorescein or rhodamine dye.
• Another preferred detectable moiety is colloidal gold.
• the colloidal gold particle is typically 40 to 80 nm in diameter.
• the colloidal gold may be attached to a labeling compound in a variety of ways.
• the linker moiety of the nucleic acid labeling compound terminates in a thiol group (-SH), and the thiol group is directly bound to colloidal gold through a dative bond.
• -SH thiol group
• it is attached indirectly, for instance through the interaction between colloidal gold conjugates of antibiotin and a biotinylated labeling compound.
• the detection of the gold labeled compound may be enhanced through the use of a silver enhancement method. See Danscher et al. J. Histotech 1993, 16, 201-207.
• the connecting groups (M) ra can serve to covalently attach the linker group (L) to the detectable moiety (Q).
• Each M group can be the same or different and can independently be any suitable structure that will not interfere with the function of the labeling compound.
• Nonlimiting examples of M groups include the following: amino alkyl, -CO(CH 2 ) 5 NH- -CO-, -CO(O)-, -CO(NH)-, -CO(CH 2 ) 5 NHCO(CH 2 ) 5 NH- -NH(CH 2 CH 2 O) k NH-, - NH(CH 2 CH 2 O) k CH 2 CH 2 NHand -CO(CH 2 ) 5 -; wherein, k is an integer from 1 to about 5, preferably k is 1 or 2; m is an integer ranging from 0 to about 5, preferably 0 to about 3.
• L vinyl
• R ⁇ is alkyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl.
• R ⁇ is C(O)R ⁇ 2 , where R 12 is a bond, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.
• the linker group L is selected to provide a linking function, which either alone or in conjunction with appropirate connecting groups (M) appropriately spaces the Q group from the Hc group (nucleotide base) at such a length and in such a configuration as to allow an appropriate assay to be performed on the Q group, but at the same time substantially preserving the ability of the nucleic acid labeling compound to act as a substrate for the appropriate enzyme, e.g., terminal transferase and/or RNA polymerase .
• M appropirate connecting groups
• R 12 is preferably a bond
• Y is OH
• Z is OH
• L is -(CH 2 ) 2 C(O)-
• Q is selected from the group consisting of a fluorescein and a biotin
• a first M is -NH(CH 2 ) 2 NH-
• a second M is -CO(CH ) 5 TSfH-, wherein m is 2.
• Specific A groups are H or H 4 O 9 P 3 -
• a specific X group is O.
• a specific Y group is H or OR 9 ,
• a specific Z group is H, N , F or ORio.
• More specific Y groups are hydrogen or OH. More specific Z groups are hydrogen or OH.
• a more specific Y group is OH.
• a more specific Z group is OH.
• R 9 groups are H, alkyl or aryl.
• a more specific Rio group is hydrogen.
• R ⁇ groups are -O-,amino, thio, carbonyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, and amino alkyl.
• Rn groups are alkyl, alkoxy, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, thio and amino alkyl.
• Rn group is C(O)R ⁇ 2 .
• R ⁇ 2 groups are a bond, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl.
• R ⁇ 2 group is a bond.
• Specific Q groups are a hapten, a fluorophore, a metal chelator, an intercalator, a luminescent moiety, a metal aggregate, and a protein.
• a more specific Q group is biotin or a fluorescent dye
• a more specific Q group is a biotin.
• a specific biotin has the structure:
• a specific M group is -NH(CH 2 ) n NH-, or -CO(CH 2 ) p NH-; wherein n is an integer from about 2 to about 10; and p is an interger from about 2 to about 10.
• a more specific M group is -NH(CH 2 ) 2 NH- , or -CO(CH 2 ) 5 NH-.
• a specific m is 2.
• a specific A group is a triphosphate group having appropriate counterions.
• A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid
• A is a triphosphate group with apporpriate counterions.
• the counterions are selected from the group consisting of are H + , Na + , Li + , K , or NH ;
• X is O;
• Y is OH;
• Z is OH;
• M is - (CH 2 ) 5 -NH- and Q is biotin having the structure:
• A is H or HUOgPs-;
• X is O;
• Y is H or OR 9 , wherein R 9 is H, alkyl or aryl;
• Z is H, N 3 , F or ORio, wherein Rio is H, alkyl or aryl;
• Q is biotin or a fluorescein; and, a first M is -NH(CH 2 ) n NH-, wherein n is an integer from about 2 to about 10, and a second M is -CO(CH 2 ) p NH-, wherein p is an interger from about two to about 10 and m is 2.
• L is vinyl
• Rn is alkyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl.
• Rn is C(O)R ⁇ 2 where R ⁇ 2 is a bond, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.
• the linker group L is selected to provide a linking function, which either alone or in conjunction with appropriate connecting group (M) ; provide appropriate spacing of the Q group from the Hc or base group at such a length and in such a configuration as to allow an appropriate assay to be performed on the Q group, but at the same time substantially preserving the ability of the nucleic acid labeling compound to act as a substrate for the appropriate enzyme, e.g., terminal transferase and/or RNA polymerase.
• M appropriate connecting group
• Hc-L-(M)m-Q groups must be chosen, in accordance with the present invention, to avoid subtantially inhibiting the ability of a nucleic acid strand incorporating such group to undergo Watson-Crick type base pairing with complementary sequences.
• -L-(M)m-Q may be any arrangements or grouping of molecules or atoms which functions to allow nucleic acids to be labeled and detected.
• R ⁇ 2 is preferably a bond
• Y is OH
• Z is OH
• L is -(CH 2 ) 2 C(O)-
• Q is selected from the group consisting of a fluorescein and a biotin
• a first M is -NH(CH 2 ) 2 NH-
• a second M is -CO(CH 2 ) 5 NH-, wherein m is 2.
• A is H or H O 9 P 3 -;
• X is O;
• Y is H or OR 9 , wherein R 9 is H, alkyl or aryl;
• Z is H, N 3 , F or ORio, wherein Rio is H, alkyl or aryl;
• L is -(CH 2 ) n C(O)-, wherein n is an integer ranging from about 1 to about 10;
• Q is biotin or a fluorescein; and, a first M is -NH(CH 2 ) n NH-, wherein n is an integer from about 2 to about 10, and a second M is -CO(CH 2 ) 5 NH- wherein m is 2.
• Y is OH; Z is OH; L is -(CH 2 ) 2 C(O)-, Q is a carboxyfluorescein; and, a first M is -NH(CH 2 ) 2 NH-, and a second M is -CO(CH 2 ) 5 NH-, wherein m is 2.
• Y is OH; Z is OH; L is -(CH 2 ) 2 C(O)-, Q is biotin; and, a first M is -NH(CH 2 ) 2 NH-, and a second M is -CO(CH 2 ) 5 NH-, wherein m is 2.
• A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid; preferably, A is a triphosphate group with apporpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH 4 +;
• X is O;
• Y is OH;
• Z is OH;
• M is -(CH 2 ) 5 -NH- and Q is biotin having the structure:
• A is H or H 4 O 9 P 3 -;
• X is O;
• Y is H or OR 9 , wherein R 9 is H, alkyl or aryl;
• Z is H, N 3 , F or ORio, wherein Rio is H, alkyl or aryl;
• Q is biotin or a fluorescein; and, a first M is
• Y is H or OH;
• Z is H or OH;
• a first M is -NH(CH 2 ) 2 NH-, and a second M is -CO(CH ) 5 NH-, wherein m is 2.
• L is vinyl
• Rn is alkyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl.
• Rn is C(O)R 12; where R 12 is a bond, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.
• the linker group L is selected to provide a linker function, which either alone or in conjunction with appropriate connecting groups (M), appropriately spaces the Q group from the Hc or base group at such a length and in such a configuration as to allow an appropriate assay to be performed on the Q group, but at the same time substantially preservers the ability of the nucleic acid labeling compound to act as a substrate for the appropriate enzyme, e.g., terminal transferase and/or RNA polymerase.
• M appropriate connecting groups
• Hc-L-(M)m-Q groups must be chosen in accordance with the present invention, to avoid subtantially inhibiting the ability of a nucleic acid strand incorporating such group to undergo Watson-Crick type base pairing with complementary sequences.
• -L-(M)m-Q may be any arrangements or gourping of molecules or atoms which functions to allow nucleic acids to be labeled and detected.
• R ⁇ 2 is preferably a bond
• Y is OH
• Z is OH
• L is -(CH 2 ) 2 C(O)-
• Q is selected from the group consisting of a fluorescein and a biotin
• a first M is -NH(CH 2 ) 2 NH-
• a second M is -CO(CH 2 ) 5 NH-, wherein m is 2.
• Nucleic acids can be isolated from a biological sample or synthesized, on a solid support or in solution for example, according to methods known to those of skill in the art. As used herein, there is no theoretical limitation on the length or source of the nucleic acid used in the labeling process. Limitations on length may, however, be imposed or suggested by the hybridization process. Exemplary methods of nucleic acid isolation and purification are described in Theory and Nucleic Acid Preparation. In Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes; P. Tijssen, Ed.; Part I; Elsevier: N.Y., 1993.
• a preferred method of isolation involves an acid guanidinium-phenol-chloroform extraction followed by oligo dT column chromotography or (dT)n magnetic bead use.
• oligo dT column chromotography or (dT)n magnetic bead use.
• the nucleic acids are increased in quantity through amplification.
• Suitable amplification methods include, but are not limited to, the following examples: polymerase chain reaction (PCR) (Innis, et al. PCR Protocols.
• nucleic acid labeling compound can be incorporated into a nucleic acid using a number of methods.
• nucleic acid sample e.g., mRNA, polyA mRNA, cDNA
• amplification product e.g., amplification product
• Methods of attaching a labeling compound to a nucleic acid include, without limitation, nick translation, 3 -end-labeling, ligation, in vitro transcription (IVT) or random priming.
• the nucleic acid is an RNA
• a labeled riboligonucleotide is ligated, for example, using an RNA ligase such asT4 RNA Ligase. See 77ze Enzymes; Uhlenbeck and Greensport, Eds.; Vol. XV, Part B, pp.
• Terminal transferase is used to add deoxy-, dideoxy- or ribonucleoside triphosphates (dNTPs, ddNTPs or NTPs), for example, where the nucleic acid is single stranded DNA.
• the labeling compound can also be incorporated at an internal position of a nucleic acid.
• PCR in the presence of a labeling compound provides an internally labeled amplification product.
• IVT in the presence of a labeling compound can provide an internally labeled nucleic acid.
• the nucleic acid to which the labeling compound is attached can be detected after hybridization with a nucleic acid probe.
• the probe can be labeled, depending upon the experimental scheme preferred by the user.
• the probe is a nucleic acid, or a modified nucleic acid, that is either attached to a solid support or is in solution. It is complementary in structure to the labeled nucleic acid with which it hybridizes.
• the solid support is of any suitable material, including polystyrene based beads and glass chips.
• the probe or target nucleic acid is attached to a glass chip, such as a GeneChip ® product (Affymetrix, Inc., Santa Clara, CA). See International Publication Nos.
• nucleic acid labeling compound Because probe hybridization is often a step in the detection of a nucleic acid, the nucleic acid labeling compound must be of a structure that does not substantially interfere with that process. The steric and electronic nature of the labeling compound, therefore, is compatible with the binding of the attached nucleic acid to a complementary structure.
• a primary goal of the invention is to provide new reagents for two particular labeling procedures: (L), 3' end labeling of fragmented, PCR-generated DNA targets with terminal deoxynucleotidyl transferase (TdT); and (ii.), template-directed internal labeling of in vitro transcription-generated RNA targets with T7 RNA polymerase (XI).
• the general approach taken was to screen various base-substituted nucleotide analogs, using a rapid and quantitative HPLC-based assay, to empirically determine which analogs were efficient substrates for the polymerase of interest.
• the analogs selected for this study were nucleotides in which the native heterocyclic base was substituted with the following: l-(imidazole-4- carboxamide), l-(l,3,6-trazine-2,4-dione), 5-(l,3-pyrimidine-2,4-dione), 3- (pyrazalo-[4,3-d] pyrimidine), l-(pyrazalo-[3,4-d]pyrimidine) and a simple carboxamide moiety. Labeled versions of promising candidate molecules were then designed and synthesized for further testing of relative incoproation efficiency and functional performance in array-based assays.
• TdT was generally tolerant of base substitutions, and that ribonucleotides were about as efficiently incorporated as 2'-deoxy, and 2', 3'-dideoxynucleotides.
• T7 was relatively intolerant of heterocyclic base substitutions with the exception of the 5-(l,3-pyrimidine-2,4- dione), i.e. the pseudo-uridine analog.
• the incorporation efficiency is expressed as the fraction of oligonucleotide that is labeled. This number is determined by dividing the peak area measured at 260 nm absorbance of the labeled oligonucleotide by the sum of the peak areas of the unlabeled and labeled oligonucleotide. ( The retention time of fluorescein-labeled dT 16 is on the order of 2 to 3 min. longer than the unlabeled dT 16 .) The error in this type of assay is about 10%.
• the IVT incorporation efficiency (the number of labeled analogs incorporated per transcript) of the Nl-fluorescein-X-5-( ⁇ -D-ribofuranosyl)-
• 2,4(l ⁇ ,3 ⁇ )-pyrimidinedione 5 '-triphosphate 42a was measured by HPLC (diode array UV detection at 260 nm and 495 nm) in an IVT amplification of a 1.24 kb transcript. See US patent application SN 09/126,645 for additional details on test methods used. Chart 1 summarizes the data obtained using different ratios of UTP/5 At a ratio of 1 :5, the incorporation and relative yield (measured relative to the yield obtained with UTP only) of transcript are optimal. This transcript was compared in a hybridization assay to transcript labeled using fluorescein.
• the labeling reaction conditions are the standard conditions used in the Affymetrix HIN-PRT GeneChip product assay (see Kozal, et al. Nature Medicine 1996, 2: 753-9.).
• Chart 2 Call accuracy ofNl-fluorescein-labeled 5-( ⁇ -D-ribofuranosyl)- 2,4(1 H, 3 H) -pyrimidinedione 5 '-triphosphate 42a.
• Pseudoisocytidine (1) (2.5g, 9mmoles) was dissolved in 40 ml dry pyridine. Acetic anhydride (8.5 ml, 90 mmoles) was added and the mixture was stirred under argon for at least 4 hours at room temperature. The reaction can be monitored by HPLC (C18 column, buffer A: 0.1M TEAA, pH 7.5; buffer B: acetonitrile; gradient: 5-95%B over 20 minutes). The pyridine was removed under vacuum and the residual oil was dissolved in 500 ml of ethyl acetate. More ethyl acetate may be added to get a clear solution since the product has limited solubility in ethyl acetate. The organic phase was washed three times with brine and dried over anhydrous Na 2 SO j filtered and the solvent removed. The white solid was recrystallized from ethyl acetate/hexane yielding 3.2 g (85%) of 2.
• the aqueous layer was back extracted with two 200 ml-portions of ethyl acetate.
• the combined organic layer was dried over anhydrous Na SO 4 , filtered and the solvent removed.
• the residue was purified by flash column chromatography on silica gel (200 ml wet gel) using ethyl acetate as the eluent affording 850mg (35%) of 3 as a white foam.
• the compound was purified by preparative HPLC: PRP-1, 30 x 250mm column; flow rate 25 ml/min; buffers: A, 0.1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes. Salts were removed with a retention time of about 4 min. and the compound eluted from 6 to 7.5 minutes. The collected fractions were pooled and the solvent removed under vacuum. The residue which contained triethylammonium acetate was co-evaporated with water several times and finally the product was precipitated from methanol/acetonitrile to afford 290mg (51%) of 5.
• Biotin-propenamide 6 Compound 5 (280 mg, 0.79mmoles) was dissolved in dry DMF (5 ml) followed by the addition of triethylamine (160mg, 220 ⁇ l, 1.58 mmoles). The pH of the solution was adjusted to 7.5 with the addition of more triethylamine, if necessary. Biotin-X-NHS ester (358 mg, 0.79 mmoles,) was then added to the mixture with stirring. After 1.5 hours the solvent volume was reduced under vacuum to about 1 ml. Caution: do no vacuum to dryness because this compound tends to aggregate and it will be difficult to redissolve.
• the compound was purified by preparative HPLC: PRP-1, 30 x 250mm column; flow rate 25 ml/min; buffers: A, 0.1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes. Salts were removed with a retention time of about 4 min. and the compound eluted from 6 to 7.5 minutes. The fractions were pooled and the solvent removed under vacuum. The residue which contained triethylammonium acetate was co-evaporated with water several times and finally the product was precipitated from methanol/acetonitrile to afford 700 mg (62%) of 4a.
• Biotin-propenamide 5a Compound 4a (102 mg, 0.286 mmoles) was co-evaporated with dry DMF twice (5 ml each) and then dissolved in dry DMF (1.5 ml) followed by the addition of triethylamine (29 mg, 40 ⁇ l, 0.286 mmoles). The pH of the solution was adjusted to 7.5 with the addition of more triethylamine, if necessary. Biotin- X-NHS ester (0.286 mmoles, 130 mg) was then added to the mixture with stirring. After 1.0 hour, the reaction was monitored by HPLC for completion. The solvent volume was reduced under vacuum to about 1 ml. Caution: do not vacuum to dryness because this compound tends to aggregate and it will be difficult to redissolve. The residual was redissolved in 5 ml water and 1 ml methanol.
• the compound was purified by preparative HPLC: PRP-1, 30 x 250mm column; flow rate 25 ml/min; buffers: A, 0.1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 11 minutes, then 0 to 95% B over 16 minutes. Fractions were collected across the peak from 19-21 min. The solvent was removed under vacuum and the residue was co-evaporated with water (30 ml) three times and methanol (50 ml) two times. The product was recrystallized from acetonitrile yielding 130mg (67%) of 5a.
• Compound 5a (130mg, 0.187 mmoles) was dried over P 2 O 5 under vacuum for 24 hours and then dissolved in trimethyl phosphate (dried over molecular sieves, 20 ml) with gentle heating to about 60 C. Once the material dissolved the solution was cooled to ambient temperature and a trap-pack (ABI Trap-pak, P#GEN 084034) was added and allowed to gently stir overnight. The solution turned into a little cloudy when chilled on ice. The trap-pack was removed and to the solution at 0°C under argon was added POCl 3 (115 mg, 70 ⁇ l, 0.748 mmoles).
• the reaction was monitored by AX-HPLC for the conversion to the monophosphate, and after 4 hours, an additional one equivalent of POCl 3 were added and the reaction was allowed to stir for 2 more hours (90% conversion).
• the mixture was then analyzed by HPLC (70% triphosphate) and purified using standard TriLink procedures on DEAE.
• the final reaction mixture may be diluted with mili Q water by a factor of 100, and then loaded on DEAE column. It is not recommended to rotovap off TEAB because the compound may be unstable under basic condition.
• TBA-PPi Aldrich, P-8533, 1.5 TBA per PPi, 1.4g, 3.1mmoles
• TBA 287 mg, 364 ⁇ l, 1.55 mmoles Co-evaporate with 5 ml dry DMF at least three times. Redissolve in 5 ml anhydrous DMF.
• TBA (1.46 ml, 3.1 mmoles). Handle the materials in a glove box filled with Ar.
• INT incorporation was determined spectrophotometrically using 260nm absorbance for the quantitation of R ⁇ A and a HABA-based colorimetric assay for quantitation of biotin for RLR-3b (Biotin- ⁇ isoCTP, vinyl-linked), RLR-2b (Biotin- ⁇ UTP, vinyl-linked) and RLR-2a (Biotin- ⁇ UTP, ethane (or saturated)- link).
• the vinyl linked analogs were both incorporated more efficiently than the saturated ethane analog as shown in Figure 7.

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