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`Case 1:12—cv—OO274—LPS Document 74-1 Filed 05/06/13 Page 1 of 43 Page|D #: 803
`
`EXHIBIT A
`
`

`
`Case 1:12-cv-00274-LPS Document 74-1 Filed 05/06/13 Page 2 of 43 PageID #: 804
`Case “2"‘”'°°27“'”"°’ 0°"””‘e“‘ ml ||ll||flI||fll||li|Ilfl|||llllll||1||lllllfillllflllfillllllllllm
`
`US006992180B1
`
`(12) Ulllted States Patent
`Engelhardt et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,992,180 B1
`Jan. 31, 2006
`
`(54) 0LIGO- OR POLYNUCLEOTIDES
`COMPRISING PHOSPHATE-MOIETY
`
`4/1981 Scherberg
`4,260,737 A
`4,358,535 A * 11/1982 Falkow et al.
`
`............... .. 435/5
`
`LABELED NUCLEOTIDES
`
`(75)
`
`Inventors: Dean Engelhardt, New York, NY (US),
`Elazar Rabbani, New York, NY (US);
`Stanley Kline, Brooklyn, NY (US);
`Janes G. Stavrianopoulos, New York,
`NY (US); Dollie Kirtikar, Elmhurst,
`NY (US)
`(73) Assignee: Enzo Life Sciences, Inc. c/o Enzo
`Biochem, Inc., Farmingdale, NY (US)
`
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`
`2507901
`
`“/1970
`(Continued)
`OTHER PUBLICATIONS
`
`M k
`t
`ac ey e a
`
`16 20 :4478-4482, 1977.*
`l., B'
`h
`'
`t
`(
`10C emls ry
`)
`(Continued)
`
`-
`( * ) Notice:
`
`-
`-
`-
`-
`Subject to any d1scla1mer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`Primary Examiner—John S. Brusca
`.
`.
`.
`(74)dAtt0rney, Agent, or FLrm—Ronald C. Fedus, Natahe
`Bog “I05
`
`(21) Appl. No.: 08/479,997
`
`(22) Filed,
`
`Jun_ 7, 1995
`
`(57)
`
`The present
`formula,
`
`ABSTRACT
`
`invention provides a nucleotide having the
`
`Related U.S. Application Data
`
`sig.PM.sM.BAs]-3
`
`(60) Continuation of application No. 08/046,004, filed on
`Apr. 9, 1993, now abandoned, which is a continuation
`of application No. 07/532,461, filed on May 31, 1990,
`now abandoned, which is a division of application
`No. 07/140,980, filed on Jan. 1, 1988, now aban-
`doned, which is a continuation of application No.
`06/674,352, filed on Nov. 21, 1984, now abandoned,
`Which is a continuation of application No. 06/391,
`440, filed on Jun. 23, 1982, now abandoned.
`
`(51)
`
`1111- Cl-
`(2006-01)
`C1 2N 15/11
`(52) U.S. Cl.
`................................. .. 536/23.1;536/25.32
`(58) Field of Classification Search .................. .. 435/6,
`435/7.1; 536/26.6, 25.31, 25.32, 23.1
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`wherein PM is a phosphate moiety, SM is a sugar moiety and
`BASE is a pyrimidine, purine or 7-deazapurine moiety. PM
`is attached to the 3‘ or the 5‘ position of the sugar moiety
`when the nucleotide is a deoxyribonucleotide and at the 2‘,
`3‘ or 5‘ position when the nucleotide is a ribonucleotide.
`BASE is attached to the 1‘ position of SM from the N1
`position when BASE is a pyrmidine or the N9 position when
`BASE is a purine or 7-deazapurine. Sig is covalently
`attached to PM directly or via a chemical linkage, and
`represents a detectable moiety covalently attached to SM
`directly or through a linkage group. Also provided are an
`oligo- or polynucleotide comprising at least one such phos-
`phate-moiety labeled nucleotide, and other compositions
`including those wherein a polypeptide is terminally ligated
`or attached to the oligo- or polynucleotide. The phosphate-
`moiety labeled nucleotide, and the oligo- or polynucleotides
`and other compositions comprising such phosphate-moiety
`labeled nucleotides, are useful as diagnostic tools for detect-
`ing analytes and as therapeutic agents.
`
`4,224,408 A *
`
`9/1980 Hung et al.
`
`................. .. 435/91
`
`116 Claims, 4 Drawing Sheets
`
`——°—’:*:'?:' Cl DNA
`
`100
`
`80
`
`60
`
`40
`
`E E
`
`C)
`
`Eg
`
`(8 20
`
`0 R0 100 200 300 400 500
`
`
`
`
`
`MICROGRAM CON A PZR REACTION VOLUME
`
`

`
`Case 1:12-cv-00274-LPS Document 74-1 Filed 05/06/13 Page 3 of 43 PageID #: 805
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`
`US 6,992,180 B1
`Page 2
`
`U.S. PATENT DOCUMENTS
`
`.............. .. 536/29
`3/1983 Gohlke et al.
`4,378,458 A *
`4,711,955 A * 12/1987 Ward et al.
`................. .. 536/29
`4,847,240 A
`7/1989 Ryser et al.
`5,212,059 A
`5/1993 Schwartz et al.
`5,242,906 A
`9/1993 Pagano et al.
`5,591,600 A
`1/1997 Ecker, et al
`5,591,720 A
`1/1997 Anderson et al.
`5,614,617 A
`3/1997 Cook et al.
`5,643,730 A
`7/1997 Banker et al.
`5,643,780 A
`7/1997 Baker
`5,652,094 A
`7/1997 Usman et al.
`5,665,710 A
`9/1997 Rahman et al.
`5,706,498 A
`1/1998 Fujimiya et al.
`5,736,294 A
`4/1998 Ecker, et al.
`5,807,677 A
`9/1998 Eigen et al.
`5,811,232 A
`9/1998 Crooke et al.
`5,874,564 A
`2/1999 Ecker, et al.
`5,891,468 A
`4/1999 Martin et al.
`5,914,230 A
`6/1999 Liu et al.
`5,953,727 A
`9/1999 Maslyn et al.
`5,966,712 A
`10/1999 Sabatini et al.
`5,980,096 A
`11/1999 Thalhammer—Reyero
`5,998,383 A
`12/1999 Wright et al.
`6,189,013 B1
`2/2001 Maslyn et al.
`6,303,297 B1
`10/2001 Lincoln et al.
`6,308,170 B1
`10/2001 Balaban
`FOREIGN PATENT DOCUMENTS
`
`EP
`GB
`JP
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`WO
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`0061761 A1
`2040943 A
`5742632
`61103824
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`
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`5/1986
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`and Nucleotides,” Annals of the New York Academy of
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`tion of Double-Stranded RNA and Virus Antigen in RNA
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`

`
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`
`US 6,992,180 B1
`Page 3
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`“Peptidyl-tRNA With A
`al.,
`et
`Kukhavova, M. K.,
`Fluorescent Label: Ribosome Substrates in Peptide Bond
`Formation,” Molecular Biology Reports 1: 397-400 (1974).
`Lynch, D. C. and Schimmel, P. R., “Effects of Abnormal
`Base Ionizations on Mg2 + Binding to Transfer Ribonucleic
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`1852-1861 (1974).
`Bergstrom, D. E. and Leonard N. J ., “Structure of the
`Borohydride Reduction Product of Photolinked 4-Thiouracil
`and Cytosine. Fluorescent Probe of Transfer Ribonucleic
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`Biochimica Et Biophysica AC1A 269: 225-236 (1972).
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`and Chang, S. H., “A Fluorescent
`Nucleoside from Glutamic Acid tRNA of Escherichia coli
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`K12,” Biochemical and Biophysical Research Communica-
`tions 46(5): 1837-1842 (1972).
`Erlanger, B. F., et al., “Nucleic Acid-Reactive Antibodies
`Specific
`for Nucleosides
`and Nucleotides,” Acta
`endocrinologic 71: 206-221 (1972).
`Yoshikami, D. and Keller, E. B., “Chemical Modification of
`the Fluorescent Base in Phenylalanine Transfer Ribonucleic
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`Rudkin G. T. and Siollar, B. D., “High Resolution Detection
`of DNA-RNA Hybrids
`in
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`munofluorescence,” Nature 265: 472-473 (1977).
`Rozovskaya, T. A., et al., “Introduction of a Fluorescent
`Label at the 3‘-OH End of DNA and the 3‘-OH End of the
`
`Growing RNA Chain,” Molekulyama Biologiya 11(3): 598-
`610 (1977).
`in
`“Recent Advances
`and Seliger, H.,
`Kossel, H.
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`Organic Natural Products 32: 297-508 (1975).
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`298-299 (1976).
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`Sugar Series; The Structural Basis of Bitterness in Sugar
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`
`

`
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`
`U.S. Patent
`
`Jan. 31, 2006
`
`Sheet 1 of 4
`
`US 6,992,180 B1
`
`F/G.
`
`7
`
`100
`
`80
`
`60
`
`40
`
`20
`
`
`
`A260PERCENTOFINPUT
`
`MICROCRAM CON A PER REACTION VOLUME
`
`

`
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`
`U.S. Patent
`
`Jan. 31, 2006
`
`Sheet 2 of 4
`
`US 6,992,180 B1
`
`DNA - BINDING TO CON A SEPHAROSE
`pH ELUTION
`
`7.8
`
`7.9
`
`8.0
`
`8.1
`
`pH
`
`1 DNA
`‘—
`
`7.2
`
`7.4
`
`TRIS-N0 CL
`7.6
`7.7
`
`PBS
`6.5
`
`50
`
`40
`
`N §
`
`3o
`
`E $
`
`CL
`
`20LAJ
`
`10
`
`
`
`PERCENTRECOV.7..
`
`ELUTION VOLUME IN mL
`
`

`
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`
`U.S. Patent
`
`Jan. 31, 2006
`
`Sheet 3 of 4
`
`US 6,992,180 B1
`
`F/G. 3,4
`
`DNA BINDING To CON A SEPHAROSE
`EFFECT ON MANNOSE
`
`PBS + MANNOSE
`
`T4 DNA
`
`
`
`3HcpmPERCENTorTOTALINPUT
`
`
`
`3HcpmPRECENTOFTOTALINPUT 5
`
`(/4 3
`
`f\) 3
`
`5
`
`10
`
`15
`
`20
`
`ELUTION VOLUME IN mil
`
`

`
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`
`U.S. Patent
`
`Jan. 31, 2006
`
`Sheet 4 of 4
`
`US 6,992,180 B1
`
`F/G.
`
`4/4
`
`CON-A SEPHAROSE
`
`BINDING OF GLUCOSYLATED DNAS
`3/8-9/82
`
`P88 6.5
`
`J
`
`8.2 TRIS
`
`5
`
`MALTOTRIOSE-LABELLED
`
`LAMBDA DNA
`
`3Hcpm7;orINPUT
`
`TRANSLATED
`
`5
`
`10
`
`15
`
`ELUTION VOLUME IN M
`
`60
`
`UNSUBSTITUTED
`LAMBDA DNA
`
`3Hcpm%OFINPUT
`
`5
`
`10
`
`15
`
`ELUTION VOLUME IN Ml
`
`

`
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`US 6,992,180 B1
`
`1
`OLIGO- OR POLYNUCLEOTIDES
`COMPRISING PHOSPHATE-MOIETY
`LABELED NUCLEOTIDES
`
`CROSS-REFERENCE TO OTHER RELATED
`APPLICATIONS
`
`This application is a continuation of U.S. patent applica-
`tion Ser. No. 08/046,004, filed on Apr. 9, 1993, now aban-
`doned, which application is a continuation of U.S. patent
`application Ser. No. 07/532,461, filed on May 31, 1990, also
`abandoned, which is a division of U.S. patent application
`Ser. No. 07/140,980, filed on Jan. 1, 1988, abandoned, which
`is a continuation of U.S. application Ser. No. 06/674,352,
`filed on Nov. 21, 1984, abandoned, which in turn was a
`continuation of U.S. application Ser. No. 06/391,440, filed
`on Jun. 23, 1982, abandoned. From the aforementioned Ser.
`No. 07/140,980,
`two divisional applications, U.S. patent
`application Ser. No. 07/532,704 (filed on Jun. 4, 1990) for
`“Base Moiety Labeled Detectable Nucleotide” and Ser. No.
`07/567,039 (filed on Aug. 13, 1990) for “Saccharide Specific
`Binding System Labeled Nucleotides” issued as U.S. Pat.
`No. 5,241,060 (Aug. 31, 1993) and U.S. Pat. No. 5,260,433
`(Nov. 9, 1993), respectively.
`
`BACKGROUND OF THE INVENTION
`
`It is known to produce nucleotides or polynucleotides
`which are radioactively labeled, such as with isotopes or
`hydrogen (3H), phosphorus (32P), carbon (14C) or iodine
`(1251). Such radioactively labeled compounds are useful to
`detect, monitor, localize and isolate nucleic acids and other
`molecules of scientific or clinical interest. Unfortunately,
`however, the use of radioactively labeled materials presents
`hazards due to radiation. Also due to the relatively short half
`life of the radioactive materials employed to label such
`compounds or materials, the resulting labeled compounds or
`materials have a corresponding relatively short shelf life.
`It has been proposed to chemically label compounds of
`interest, such as nucleotides and polynucleotides, so as to
`overcome or avoid the hazards and difficulties associated
`
`with such compounds or materials when radioactively
`labeled. In the article by P. R. Langer, A. A. Waldrop and D.
`C. Ward entitled “Enzymatic Synthesis of Biotin-Labeled
`Polynucleotides: Novel Nucleic Acid Affinity Probes”, in
`Proc. Natl. Acad. Sci., USA, Vol. 78, No. 11, pp. 6633-6637,
`November, 1981, there are described analogs of dUTP and
`UTP that contain a biotin molecule bound to the C-5 position
`of the pyrimidine ring through an alkylamine linker arm.
`The biotin-labeled nucleotides are efficient substrates for a
`
`variety of DNA and RNA polymerases in vitro. Polynucle-
`otides containing low levels of biotin substitution (50 mol-
`ecules or fewer per kilobase) have denaturation, reassocia-
`tion and hybridization characteristics similar to those of
`unsubstituted controls. Biotin-labeled polynucleotides, both
`single and double stranded, are selectively and quantita-
`tively retained on avidin-Sepharose, even after extensive
`washing with 8M urea, 6M guanidine hydrochloride or 99%
`formamide. In addition, biotin-labeled nucleotides can be
`selectively immunoprecipitated in the presence of antibiotin
`antibody and Staphylococcus aurea, Protein A. These
`unique features of biotin-labeled polynucleotides suggest
`that they are useful affinity probes for the detection and
`
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`isolation of specific DNA and RNA sequences. It is indicated
`in the article that the subject matter of the article is com-
`prised in a pending U.S. patent application.
`
`The disclosure of this article and above-referred pending
`patent application are herein incorporated and made part of
`this disclosure.
`
`The patent application referred to in the above-identified
`article is U.S. patent application Ser. No. 255,223 filed Apr.
`17, 1981. Ser. No. 06/255,223 was abandoned in favor of
`continuation application, U.S. patent application Ser. No.
`06/496,915, filed on May 23, 1983, now U.S. Pat. No.
`4,711,955. A related divisional application of the aforemen-
`tioned Ser. No. 06/496,915 was filed as U.S. patent appli-
`cation Ser. No. 07/130,070 (on Dec. 8, 1987), and has since
`issued on Jul. 12, 1994 as U.S. Pat. No. 5,328,824. Two
`related continuation applications of the aforementioned Ser.
`No. 07/130,070 were filed on Feb. 26, 1992 (as Ser. No.
`07/841,910) and on May 20, 1992 as (Ser. No. 07/886,660).
`The former, Ser. No. 07/841,910, has been allowed, and the
`latter, Ser. No. 07/886,660, issued as U.S. Pat. No. 5,449,767
`on Sep. 12, 1995. Therefore, the disclosures of all three
`aforementioned U.S. Pat. Nos. 4,711,955, 5,328,824 and
`5,449,767 are herein incorporated by reference and made
`part of the instant disclosure. The disclosures of this pending
`U.S. patent application Ser. No. 255,223 are herein incor-
`porated and made part of this disclosure. In the above-
`identified pending U.S. patent application the subject matter
`of the above-identified article is disclosed and additionally it
`is disclosed that compounds having the structure:
`
`
`
`wherein B represents a purine, deazapurine, or pyrimidine
`moiety covalently bonded to the C1’-position of the sugar
`moiety, provided that when B is purine or 7-deazapurine, it
`is attached at the N9-position of the purine or deazapurine,
`and when B is pyrimidine, it is attached at the N1-position;
`wherein A represents a moiety consisting of at least three
`carbon atoms which is capable of forming a detectable
`complex with a polypeptide when the compound is incor-
`porated into a double-stranded ribonucleic acid, deoxyribo-
`nucleic acid duplex, or DNA-RNA hybrid;
`wherein the dotted line represents a chemical linkage
`joining B and A, provided that if B is purine the linkage is
`attached to the 8-position of the purine, if B is 7-deazapu-
`rine, the linkage is attached to the 7-position of the dea-
`zapurine, and if B is pyrimidine, the linkage is attached to
`the 5-position of the pyrimidine; and
`wherein each of x, y, and z represents
`
`Ho—, Ho—P—o—,
`
`OH
`
`

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`3
`
`-continued
`
`ll
`ll
`HO—I|’—O—I|>—O—,
`H
`OH
`
`or
`
`ll
`ll
`ll
`HO—I|>—O—I|’—O—I|’—O—,
`OH
`OH
`OH
`
`US 6,992,180 B1
`
`4
`
`5
`
`10
`
`
`
`(b) reacting said mercurated compound with a chemical
`moiety reactive with the —Hg+ portion of said mercu-
`rated compound and represented by the formula ***N,
`said reaction being carried out in an aqueous solvent
`
`end 1“ the Presence Of K2PdC14 under Sultable e0nd1'
`tions so as to form a compound having the structure:
`
`
`
`B"'N
`
`H
`
`z
`
`X—CH2
`
`H
`
`y
`
`wherein N is a reactive terminal functional group or is
`A; and
`
`(c) recovering said compound as said modified nucleotide
`when N is A, or when N is a reactive terminal group,
`reacting said compound with a compound having the
`structure M-A, wherein M represents a functional
`group reactive with N in an aqueous solvent under
`suitable conditions, so as to form said modified nucle-
`otide, which is then recovered.
`
`are widely useful as probes in biomedical research and 15
`recombinant DNAteChn010gy.
`Particularly useful are compounds encompassed within
`this structure which additionally have one or more of the
`following characteristics: A is non-aromatic; Ais at least C5;
`the chemical linkage joining B and A includes an ot-olefinic 20
`bond; A is biotin or iminobiotin; and B is a pyrimidine or
`7-deazapurine.
`These compounds may be prepared by a process which
`involves:
`
`25
`
`30
`
`(a) reacting a compound having the structure:
`
`B
`
`O
`
`x—CH2
`
`with a mercuric salt in a suitable solvent under suitable 35
`
`conditions so as to form a rneronrated compound hav-
`ing the structure:
`
`This invention also provides compounds having the struc-
`ture:
`
`O
`
`Ho—i>--- -O—CH2
`OH
`
`

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`US 6,992,180 B1
`
`5
`wherein each of B, B‘, and B“ represents a purine,
`7-deazapurine, or pyrimidine moiety covalently bonded to
`the C1’-position of the sugar moiety, provided that whenever
`B, B‘, or B“ is purine or 7-deazapurine, it is attached at the
`N9-position of the purine or 7-deazapurine, and whenever B,
`B‘, or B“ is pyrimidine, it is attached at the N1-position;
`wherein A represents a moiety consisting of at least three
`carbon atoms which is capable of forming a detectable
`complex with a polypeptide when the compound is incor-
`porated into a double-stranded duplex formed with a
`complementary ribonucleic or deoxyribonucleic acid mol-
`ecule.
`
`wherein the dotted line represents a chemical linkage
`joining B and A, provided that if B is purine the linkage is
`attached to the 8-position of the purine, if B is 7-deazapu-
`rine, the linkage is attached to the 7-position of the dea-
`zapurine, and if B is pyrimidine, the linkage is attached to
`the 5-position of the pyrimidine;
`wherein z represents H— or HO—; and
`wherein m and n represent integers from 0 up to about
`100,000.
`These compounds can be prepared by enzymatic poly-
`merization of a mixture of nucleotides which include the
`
`modified nucleotides of this invention. Alternatively, nucle-
`otides present in oligo- or polynucleotides may be modified
`using chemical methods.
`Nucleotides modified in accordance with the practices of
`this invention and oligo- and polynucleotides into which the
`modified nucleotides have been incorporated may be used as
`probes in biomedical
`research, clinical diagnosis, and
`recombinant DNA technology. These various utilities are
`based upon the ability of the molecules to form stable
`complexes with polypeptides which in turn can be detected,
`either by means of properties inherent in the polypeptide or
`by means of detectable moieties which are attached to, or
`which interact with, the polypeptide.
`Some uses include detecting and identifying nucleic acid-
`containing etiological agents, e.g. bacteria and viruses;
`screening bacteria for antibiotic resistance; diagnosing
`genetic disorders, e.g. thalassemia and sickle cell anemia;
`chromosomal karyotyping; and identifying tumor cells.
`Several essential criteria must be satisfied in order for a
`
`modified nucleotide to be generally suitable as a substitute
`for a radioactively-labeled form of a naturally occurring
`nucleotide. First, the modified compound must contain a
`substituent or probe that is unique, i.e., not normally found
`associated with nucleotides or polynucleotides. Second, the
`probe must react specifically with chemical or biological
`reagents to provide a sensitive detection system. Third, the
`analogs must be relatively efficient substrates for commonly
`studied nucleic acid enzymes, since numerous practical
`applications
`require that
`the analog be enzymatically
`metabolized, e.g., the analogs must function as substrates for
`nucleic acid polymerases. For this purpose, probe moieties
`should not be placed on ring positions that sterically, or
`otherwise, interfere with the normal Watson-Crick hydrogen
`bonding potential of the bases. Otherwise, the substituents
`will yield compounds that are inactive as polymerase sub-
`strates. Substitution at ring positions that alter the normal
`“anti” nucleoside conformation also must be avoided since
`
`such conformational changes usually render nucleotide
`derivatives unacceptable as polymerase substrates. Nor-
`mally, such considerations limit substitution positions to the
`5-position of a pyrimidine and the 7-position of a purine or
`a 7-diazapurine.
`Fourth, the detection system should be capable of inter-
`acting with probe substituents incorporated into both single-
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`stranded and double-stranded polynucleotides in order to be
`compatible with nucleic acid hybridization methodologies.
`To satisfy this criterion, it is preferable that the probe moiety
`be attached to the purine or pyrimidine through a chemical
`linkage or “linker arm” so that it can readily interact with
`antibodies, other detector proteins, or chemical reagents.
`Fifth, the physical and biochemcial properties of poly-
`nucleotides containing small numbers of probe substituents
`should not be significantly altered so that current procedures
`using radioactive hybridization probes need not be exten-
`sively modified. This criterion must be satisfied whether the
`probe is introduced by enzymatic or direct chemical means.
`Finally, the linkage that attaches the probe moiety should
`withstand all experimental conditions to which normal
`nucleotides and polynucleotides are routinely subjected,
`e.g., extended hybridization times at elevated temperatures,
`phenol and organic solvent extraction, electrophoresis, etc.
`All of these criteria are satisfied by the modified nucle-
`otides described herein.
`These modified nucleotides have the structure:
`
`
`
`wherein B represents a purine, 7-deazapurine, or pyrimi-
`dine moiety covalently bonded to the C1’-position of the
`sugar moiety, provided that when B is purine or 7-deazapu-
`rine,
`it
`is attached at
`the N9-position of the purine or
`7-deazapurine, and when B is pyrimidine, it is attached at the
`N1-position;
`wherein A represents a moiety consisting of at least three
`carbon atoms which is capable of forming a detectable
`complex with a polypeptide when the compound is incor-
`porated into a double-stranded ribonucleic acid, deoxyribo-
`nucleic acid duplex, or DNA-RNA hybrid;
`wherein the dotted line represents a linkage group joining
`B and A, provided that if B is purine the linkage is attached
`to the 8-position of the purine, if B is 7-deazapurine, the
`linkage is attached to the 7-position of the deazapurine, and
`if B is pyrimidine, the linkage is attached to the 5-position
`of the pyrimidine; and
`wherein each of x, y and z represents
`
`H—,
`
`HO—,
`O
`
`O
`
`ll
`HO—I|>—O—,
`OH
`
`II
`II
`HO—I|>—O—I|>—O—,
`OH
`OH
`O
`O
`O
`
`or
`
`II
`II
`II
`HO—I|’—O—I|>—O—1|>—O—
`OH
`OH
`OH
`
`These compounds are widely useful as probes in biomedical
`research and recombinant DNA technology.
`Although in principal all compounds encompassed within
`this structural formula may be prepared and used in accor-
`
`

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`US 6,992,180 B1
`
`7
`dance with the practices of this invention, certain of the
`compounds are more readily prepared or used or both, and
`therefore are presently preferred.
`Thus, although purines, pyrimidines and 7-deazapurines
`are in principal useful, pyrimidines and 7-deazapurines are
`preferred since purine substitution at the 8-position tends to
`render the nucleotides ineffective as polymerase substrates.
`Thus, although modified purines are useful
`in certain
`respects, they are not as generally useful as pyrimidines and
`7-deazapurines. Moreover, pyrimidines and 7-deazapurines
`useful in this invention must not be naturally substituted at
`the 5- or 7-positions, respectively. As a result, certain bases
`such as thymine, 5-methylcytosine, and 5-hydroxymethyl-
`cytosine are not useful. Presently preferred bases are
`cytosine, uracil, deazaadenine and deazaguanine.
`A may be any moiety which has at least three carbon
`atoms and is capable of forming a d