//{>'
`/SJJ
`
`19936 TFM
`
`',' 4
`·METHbDS AND STRUCTURES EMPLOYING NON-RADIOACTIVE
`CHEMICALLY-LABELED POLYNUCLEOTIDE PROBES
`
`5
`
`BACKGROUND OF THE DISCLOSURE
`
`In the determination of the presence or the identity of
`genetic material, such as DNA genetic material, it has
`been proposed to denature the genetic material to form
`single-stranded DNA or single-stranded genetic material.
`The ~ingle-stranded genetic material is then fixed to a
`solid support and contacted with a probe, such as a DNA
`probe, havin~ in its make up bases complementary to the
`make up of the fixed genetic material to be identified
`and/or determined. The contacting of the single-stranded
`genetic material along with the single-stranded probe is
`carried out under conditions to effect hybridization of
`the genetic material to be determined or identified and
`the probe.
`
`Radioactively-labeled probes, such as radioactively(cid:173)
`labeled single-stranded DNA probes, have been employed.
`U.S. Patent 4,358,53S discloses a methcd of identifying a
`pathogen p+esent in the clinical sample by denaturing the
`genetic material present in the clinical sample to form -
`single-stranded genetic material thereof and to fix the.
`resulting single-stranded genetic material characterizing
`the pathogen to an inert support or surface. The thus-.
`fixed single-stranded genetic material characterizing or
`identifying the pathogen is brought into contact with a
`radioactive single-stranded probe under hybridizing con(cid:173)
`ditions to effect duplex form or double-strand formation
`of the genetic material derived from the pathogen and the
`probe. The presence of the resulting formed duplex .
`between the probe and the pathogen genetic material would
`then be detected and would confirm the presence and/or
`identity of the pathogen.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Page 1 of 108
`
`HOLOGIC EXHIBIT 1004
`Hologic v. Enzo
`
`

`
`-2-
`
`The disadvantages of employing a radioactiveiy-labeled
`probe, such as a radioactively-labeled DNA probe, for the
`identification of genetic material are well known to those
`skilled in the art. Such disadvantages include not only
`the precautions and hazards involved in handling the
`radioactive material but also the short life of such
`radioactive material and expense in connection with the
`handling and use of such radioactively-labeled DNA probes .
`•
`It is known to chemically-label nucleotides and poly(cid:173)
`nucleotides to avoid the hazards and/or difficulties·
`associated when such compounds are radioactively-labeled.
`For example in the article by P.R. Langer, A.A. Waldrop
`and D.C. Ward entitled "Enzymatic Synthesis of Biotin-
`Label~d 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 allyl-
`amine linker arm. The biotin-labeled nucleotides are
`efficient substrates for a variety of DNA and RNA polymer(cid:173)
`ases in vitro. Polynucleotides containing low levels of
`biotin substitution (50 molecules or fewer per kilobaseL.
`have denaturation, reassociation and hybridization charac-
`teristics similar to those of unsubstituted controls.
`Biotin-labeled polynucleotides, both single and double
`stranded, are selectively and quantitatively retained on
`avidin-Sepharose, even after extensive washing with
`In
`8M urea, 6M guanidine hydrochloride or 99% formamide.
`addition, biotin-labeled nucleotides can be selectively
`immunoprecipitated in the presence of antibiotin antibody
`and Staphylococcus ~, Protein A. These unique
`features of biotin-labeled polynucleotides suggest that
`they are useful affinity probes for the detection and
`isolation of specific DNA and RNA sequences.
`It is
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`/i - /
`
`Page 2 of 108
`
`

`
`-3-
`
`indicated in the article that the subject matter of the
`article is comprised in a pending U.S. patent application.
`
`5
`
`Compounds or nucleotides have also been prepared which
`can be incorporated into DNA, such as double-stranded
`DNA, and which are useful for the preparation of non(cid:173)
`radioactive chemically-labeled DNA probes.
`see, for
`example, co-pending U.S. patent application Serial No.
`2~~3, filed April 17, 1981, in which the subject
`10 matter of the above-identified article is disclosed; ~nd
`additionally it is disclosed that compounds having the
`structure:
`
`B • • • A
`
`15
`
`x-<:~E:·2
`o
`a
`a.
`a
`
`Fi
`I
`
`y
`
`z
`
`wherein B represents a purine, deazapurine, or pyrimidine
`20 moiety covalently bonded to the cl'-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 deaza(cid:173)
`purine, and when B is pyrimidine, it is attached at the~·
`NLposition;
`
`25
`
`30
`
`35
`
`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 incorporated
`into a double-stranded ribonucleic acid; deoxyribonucleic
`acid duplex, or DNA-RNA hybrid;
`
`wherein the dQtted line represents a chemical linkage
`joining B and A, provided that if B is purine, the linkage
`is attached to the 9-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 att~ched to the 5-position of the pyrimidine; and
`
`Page 3 of 108
`
`

`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`-4-
`
`wherein each of x, y and z represents
`
`0
`0
`0
`0
`0
`0
`II
`II
`II
`II
`II
`II
`-o-, HO-P-0-?-0-, or H0-?-0-P-0-P-0-
`H-, HO-, HO-P
`1
`1
`I
`I
`I
`I
`OH
`OH
`OH
`OH
`OH OH
`
`I
`
`are widely useful as probes in biomedical research and
`recombinant DNA technology .
`•
`Particularly useful are compounds encompassed within
`this structure which additionally have one or more of
`the following characteristics: A is non-aromatic; A
`is at least C5; the chemical linkage joining B and A.
`includes an·a-olefinic bond; A is biotin or iminobiotin;
`and B is a pyrim~dine pr 7-deazapurine.
`
`u.s. patent application Serial No. 255,223 also discloses
`compounds having the structure:
`
`~
`HO-P
`I
`OH
`
`a' OPl
`
`H
`
`~
`0 -P -
`ba
`
`m
`
`?
`o---P
`I
`OH
`
`a"
`
`~
`o---f-- -oH
`OH
`
`n
`
`.... ....(cid:173)
`
`',::?
`
`Page 4 of 108
`
`

`
`-
`
`-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', orB" is purine or 7-deazapurine, it is attached at
`the NLposition of the p·urine or 7-deazapurine, and
`whenever B, B', or B" is pyrimidine, it is attached at the
`NLposition;
`
`whe~ein 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 incorporated
`into a double-stranded duplex formed with a complementary
`ribonucleic or deoxyribonucleic acid molecule;
`
`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-deazapurine, the linkage is attached to the a-position
`of the deazapurine, and if B is pyrimidine, the linkage
`is attached to the 5-position of the pyrimidine;
`
`5
`
`10
`
`15
`
`20
`
`wherein z represents H- or HO-; and
`
`wherein m and n represent integers from 0 up ·to about
`100,000.
`
`25
`
`These compounds can be prepared by enzymatic polymeriza(cid:173)
`tion of a mixture of nucleotides which include the modified
`nucleotides of this invention. Alternatively, nucleotides
`present in oligo- or polynucleotides may be modified
`using chemical methods.
`
`The chemically-labeled or modified nucleotides described
`in the above-referred ~ article and in U.S. patent
`application Serial No. 255,223, as indicated hereinabove
`is the structure:
`
`30
`
`35
`
`?
`
`Page 5 of 108
`
`

`
`·-
`
`-6-
`
`B • • • A
`
`x-c~2 o
`
`H
`
`H
`
`H H
`·.
`y
`
`z
`
`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-deaza(cid:173)
`purine, it is attached at the N9-position of the purine or
`7-deazapurine, and when B is pyrimidine, it is attached at
`the NLposition;
`
`5
`
`10
`
`15 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 incorpor(cid:173)
`ated into a double-stranded ribonucleic acid, deoxyribo(cid:173)
`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-deaz·a(cid:173)
`purine, the linkage is attached to the ?-position of the
`deazapurine, and if B is pyrimidine, the linkage is
`attached to the 5-position of the pyrimidine; and
`
`20
`
`25
`
`30
`
`wherein each of x, y and z represents
`0
`0
`0
`II
`rt
`1\
`HO-l?-0-
`HO-l?-0-l?-0-,
`1
`I
`\
`OH OH
`OH
`
`B-, BO-,
`
`I
`
`Ot:.
`
`0
`0
`0
`II
`II
`II
`HO-l?-0-P-0-P-0-.
`l
`I.
`I
`OH OH OH
`
`These compounds are widely useful as probes in biomedical
`research and recombinant DNA technology.
`
`Page 6 of 108
`
`

`
`-7-
`
`Although in principal, all compounds encompassed within
`this structural formula may be prepared and used in accor(cid:173)
`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 tn 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, respec(cid:173)
`tively. As a result, certain bases, such as thymine,
`5-methylcytosine, and 5-hydroxymethylcytosine, 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 detectable complex with a
`polypeptide when the modified nucleotide is incorporated
`into a double-stranded duplex containing either deoxy-
`ribonucleic or ribonucleic aicd.
`
`A therefore may be any ligand which possesses these prop(cid:173)
`erties, including haptens which are only immunogenic
`when attached to a suitable carrier, but are capable
`of interacting witn appropriate antibodies to produce
`complexes. Examples of moieties which are useful include:
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`C/
`/
`
`Page 7 of 108
`
`

`
`-8-
`
`s
`-C-(CHz) 4~ ·
`y
`
`II.
`0
`
`HN
`
`NH
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`0
`
`•
`
`-c-o~
`~\-
`
`-C-CH2-CH2C-O-·
`II
`~
`0
`0
`
`I
`
`-C-(CH )
`II
`2 4
`0
`
`(I and
`
`Of these, the preferred A moieties are ~iotin and imino(cid:173)
`biotin.
`
`Page 8 of 108
`
`

`
`., _, ... ---~-.... ,.,. .. !"'Old--
`
`-9-
`
`S
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Mcreover, since aromatic moieties tend to intercalate into
`a base-paired helical structure, it is preferred that the
`moiety A be nonaromatic. Also, since smaller moieties may
`not permit sufficient molecular interaction with poly-
`peptides, it is preferred that A be at least c 5 so that
`sufficient interaction can occur to permit formation of
`stable complexes. Biotin and iminobiotin satisfy both of
`•
`these criteria.
`
`The linkage or group joining moiety A to base B may in(cid:173)
`clude any of the well known bonds, including carbon-carbon.
`single bonds, carbon-carbon double bonds, carbon-nitrogen
`single bonds, or carbon-oxygen single bonds. However, it
`is generally preferred that the chemical linkage include
`an olefinic bond at the a-position relative to B. The
`presence of such an a-olefinic bond serves to nold the
`moiety A away from the base when the base is paired with
`another in the well known double-helix configuration.
`This permits interaction with polypeptide to occur more
`readily, thereby facilitating complex formation. More(cid:173)
`over, single bonds with greater rotational freedom may not
`always hold the moiety sufficiently apart from the helix
`to permit recognition by and complex formation with poly(cid:173)
`peptide.
`
`It is even more preferred that the chemical linkage group
`be derived from a primary amine, and have the structure
`-cH 2-NH-, since such linkages are easily formed utilizing
`any of the well known amine modification reactions. Ex-
`amples of preferred linkages derived from allylamine and
`allyl-(3-amino-2-hydroxy-1-propyl) ether groups have the
`formu~?e -CH=CH-CH2-NH- and -CH=CH-CH2-0-CH2-CH-CH2-NH-,
`.
`I
`OH
`
`respectively.
`
`./
`
`Page 9 of 108
`
`

`
`----·---
`
`-10-
`
`. Although these linkages are preferred, others can be used,
`including particularly olefin lihkage arms with other
`modifiable functionalities such as thiol, carboxylic acid,
`and epoxide functionalities.
`
`The linkage groups are attached at specific positions,
`namely, the 5-position of a pyrimidine, the a-position of
`a purine, or the 7-position of a deazapurine. As in(cid:173)
`dicated previously~ substitution at the 8-position of a
`purine does not produce a modified nucleotide which is
`•
`useful in all the methods discussed herein.· It may be
`that the ?-position of a purine, which is occupied by a
`nitrogen atom, could be the point of' linkage attachment.
`However, the chemical substitution methods employed to
`date and discussed herein are not suitable for this
`purpose.
`
`The letters x, y, and z represent groups attached to
`the 5', 3' and 2' positions of the sugar moiety. They may
`be any of
`
`H-, HO-,
`
`0
`0
`0
`II
`11
`II
`HO-P-0-, HO-P-0-P-0-
`I
`I
`I
`OH
`OH OH
`
`I
`
`or
`
`0
`0
`0
`II
`II
`II
`HO-P-0-P-P-O-.
`I
`l
`I
`OH OH OH
`
`Although conceivable, it is unlikely that all of x, y, and
`z will simultaneously be the same. More likely, at least
`one of x, y, and z will be a phosphate-containing group,
`either mono-, di-, or tri-phosphate, and at least one will
`be HO- or H-. As will be readily appreciated, the most
`likely identity of z will be HO- or H-
`indicating ribo(cid:173)
`nucleotide or deoxyribonucleotide, respectively. Examples
`of such nucleotides include 5'-ribonucleoside monophos(cid:173)
`phates, 5'-ribonucleoside diphosphates, 5'-deoxyribo(cid:173)
`nucleoside triphosphates, 5'p-ribonucleoside-3'p, and 5'p-
`deoxyribonucleoside-3'p. More specific examples include
`
`modified nucleotides of this type in which A is biotin or
`iminobiotin, the chemical linkage is
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Page 10 of 108
`
`

`
`-11-
`
`-CH=CH-CHz- NH- or -CH=CH-CHz-C-CHz ... yH-CHz- NH-
`OH
`
`,
`
`and B is uracil or cytosine.
`
`5
`
`10
`
`The general synthetic approach ado~ted for introducing the
`linker arm and probe moiety cnto the base is discussed
`thereinabove.
`(See especially, J.L. Ruth and D.E.
`Be!gstrom, and M.K. Ogawa, J. Amer. Chern. Soc. 100, 8106,
`1978; and C.F. Bigge, P. Kalaritis, J.R. Deck and M.P.
`Mertes, J. Amer. Chern. Soc. 102, 2033, 1980.) However,
`the olefin substituents employed herein have not been used
`previously. To facilitate attachment of probe moiety A,
`it has been found particularly desirable to employ olefins
`15 which primary amine functional. groups, such as allylamine
`[AA] or allyl-(3-amino-2-hydroxy-1-propyl) ether [NAGE],
`which permit probe attachmen~ by standard amine modifi(cid:173)
`cation reactions, such as,
`
`20
`
`25
`
`30
`
`35
`
`NH
`+
`fjffi2
`,, 2
`-CHzNH 2 + R-C-OR ~ -CHzNHCR
`Imidate
`
`0
`II
`-CHzNHCR
`0.~
`
`0
`II
`R-C
`\
`I
`R-C
`II
`0
`Anhydride
`
`o.
`
`¢Nc,t -+ ~cH2m.K,
`
`.
`0
`NHS-ester (N-hydroxysuccinimide)
`
`Page 11 of 108
`
`

`
`-12-
`
`s
`II
`-CHzNHz + R-i'i=C=S~-CHzNHCNHR
`Isothiocyanat:e
`
`•
`
`Epoxide
`
`Because of ease of preparation, it has been found prefer(cid:173)
`able to use NHS-esters for probe addition. However,
`olefin linker arms with other modifiable functional groups,
`such as thiols, carboxylic acids, epoxides, and the like,
`can also be employed. Furthermore, both linker arm and
`probe can be added in a single-step if deemed desirable.
`
`Having the biotin probe directly attached to the nucleo-
`tide derivatives that are capable of functioning as enzyme
`substrates offers considerable versatility, both in th~.
`experi~ental protocols that can be performed and in the
`detection me~hods (microscopic and non-microscopic) that
`can be utilized for analysis. For example, biotin nucleo-
`tides can be introduced into polynucleotides which are in
`the process of being synthesized by cells or crude cell
`extracts, thus making it possible to detect and/or isolate
`nascent (growing) polynucleotide chains. Such a procedure
`is impossible to do by any direct chemical modification
`methcd. Further~ore, enzymes can be used as reagent~ for
`introducing probes such as biotin into highly selective or
`site-specific locations in polynucleotides; the chemical
`synthesis of similar probe-modified products would be
`extremely,difficult to achieve at best.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`) {.(
`,
`
`'
`
`Page 12 of 108
`
`

`
`...,.,...,.........._....,. ___ _
`
`-13-
`
`Further,. there is disclosed i.n copending, co-as singed U.S.
`patent appLication Serial No. 3'9i,<{4,o-, filed June 23,
`1982, modified non-radioacti~~ c~emical1y-labeled nucleo(cid:173)
`tides wherein the nucleotides modified such as at the 5-
`position of pyrimidine or the ?-position of purine,
`preparatory for the preparation of nucleotide probes
`therefrom suitable for attachment to or incorporation into
`DNA~i other n~cleic acid materials.
`In the preparation
`of such modified nucleotides, the nucleotides, i.e. nucleic
`acids, preferably are modified in a non-disruptive manner
`such that the resulting modified nuclectides are capable
`of incorporation into nucleic acids and once incorporated
`in nucleic acids, the modified nucleotides do not signifi(cid:173)
`cantly interfere with the formation or stabilization of
`the double helix formed of the resulting nucleic acids
`containing the modified nucleotides. The non-disruptive
`modification of nucleotides and nucleic acids incorpora(cid:173)
`ting such modified nucleotides is in contrast with those
`modifications of nucleotides which are characterized as a
`di~ruptive modification in the sense that the resulting
`disruptively modified nucleotides and nucleic acids
`containing the same block proper double helix formation.
`In the practices of this invention, the nucleotides are
`desirably modified at the 5-position of the pyrimidine or
`the ?-position of the purine. The nucleotides so modified
`are non-disruptively modified and nucleic acids containing
`such nucleotides are capable of forming a double helix
`arrangement.
`
`Broadly, in another aspect of the practices of this in-
`vention, various methods are useful for the tagging or
`labeling of DNA in a non-disruptive manner. For example,
`biotin is added on the end of a DNA or RNA molecule. The
`addition of biotin is accomplished by addition of a
`ribonucleotide. The 3' ,4' vicinal hydroxyl groups are
`oxidized by periodate oxidation and then reduced by a
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Page 13 of 108
`
`

`
`-14-
`
`borohydride in the presence of biotin hydrazide. Alter(cid:173)
`natively, carbodiimide can also be used to couple biotin
`to the aldehyde group.
`
`5 Another technique for tagging nucleic acid material such
`as DNA or RNA involves the addition of a large marker to
`the end of a DNA or RNA molecule. One example of this
`tec~nique is the addition of a molecule, e.g. lysyl(cid:173)
`glycine, where the amino groups are tagged with biotin.
`10 Another example would be to follow the procedure set forth
`hereinabove but employing carbodiimide as the cross(cid:173)
`linking agent. Still another example of this technique
`would be to produce a biotinylated dA:dU double helical
`polymer and to ligate this polymer to the probe prepared
`in accordance with this invention.
`
`.
`
`15
`
`20
`
`Another technique for tagging DNA in a non-disruptive
`manner involves the isolation of dPyrTP having a putricine
`or spermidine on the 5-position from PS16 or phage-
`infected cells.
`If desired, dPyrTP is made from phage DNA
`and phosphorylated to dPyrTP followed by modification of
`the polyamine side chain by means of standard nucleophilic
`reagent NHS-biotin.
`
`25 Another technique for tagging DNA in a non-disruptive
`manner involves the addition of glucose to 5-hydroxy(cid:173)
`methylcytosine (5 HMC)
`in DNA using T4 phage glycosylating
`enzymes followed by screening by means of a lectin-based
`assay.
`
`30
`
`35
`
`Still another method for tagging DNA in a non-disruptive
`manner involves 5-HMC-triphosphate made from the hydro(cid:173)
`lysis of T4-DNA followed by phosphorylation of the 5HMCMP
`to 5 MMCTP..
`5 HMCTP is then incorporated into DNA using
`polymerase I. Thus, any DNA can be modified to have non(cid:173)
`disruptively incorporated therein 5 HMC.
`
`) /'
`
`I
`
`(
`
`..• •r
`
`Page 14 of 108
`
`

`
`-15-
`
`A method for tagging DNA in a mildly disruptive manner
`involves reacting nucleic acids in the double helical form
`with alkylating reagents as for example benz(o)pyrene diol
`epoxide or.aflatoxin. Under appropriate conditions of the
`N2 group of guanine, the N4 group of adenosine or the N4
`group of cytosine are alkylated. These modified nucleo(cid:173)
`tides can be used as linking arms for the addition of a
`reporter molecule such as biotin .
`•
`
`These specially modified nucleotides suitable as non(cid:173)
`radioactive chemical labels for DNA probes or DNA material
`as described in u.s. patent application Serial No. 391,440,
`can be broadly characterized and described as phosphoric
`acid P moiety, a sugar or monosaccharide S moiety, a base
`B moiety, a purine or a pyrimidine and a signalling
`chemical moiety Sig covalently attached thereto, either to
`the P, S or B moiety.
`
`5
`
`10
`
`15
`
`Of special interest are those nucleotides having a general
`formula
`
`20
`
`P - S - B - Sig
`wherein P is the phosphoric acid moiety including mono-,
`di-, tri-, or tetraphosphate, S the sugar or monosacchar~de
`moiety, B the base moiety, either a purine or a pyrimidine.
`The phosphoric acid moiety P is attached at the 3' and/or
`the 5' position of the S moiety when the nucleotide is a
`deoxyribonucleotide and at the. 2', 3' and/or 5' position
`when the r.ucleotide is a ribonucleotide. The base B moiety
`is attached from the N1 positior. of the N9 position to the
`1' position bf the S moiety when the base moiety is a
`pyrimidine or a purine, resp~ctively. The Sig moiety is
`covalently attached to the B moiety of the nucleotide and
`when so attached is capable of signalling itself or makes
`itself self-detecting or its presence known and desirably
`or preferably permits the incorporation of the resulting
`nucleotide P - s - B - Sig into or to form a double(cid:173)
`stranded helical DNA or RN/•. or DNA-RNA hybrid and/or to be
`detectable thereon.
`
`25
`
`30
`
`35
`
`Page 15 of 108
`
`

`
`-16-
`
`Another special nucleotide in accordence with this
`invention ·is characterized by the general formula:
`Sig
`
`P -.s- B
`
`Such nucleotides in accordance with this invention
`• would be characterized as ribonucleotides. The phos-
`phoric acid m~iety is attached at the 2', 3' and/or s:
`position of the sugar S moiety and the base B being
`attached from the Nl position or the N9 position to the
`1' position of the sugar S moiety when said base is a
`r
`pyrimidine or a purine, resp~ctively. The Sig chemical
`moiety is covalently attached to the sugar S moiety is
`capabie of signalling i~self or making itself self(cid:173)
`detecting or its presence known and preferably permits
`the incorporation of the ribonucleotide into its
`corresponding double~stranced RNA or a DNA-RNA hybrid.
`
`Sig
`
`Such nucleotides P - S - B desirably have the Sig
`chemical moiety attached to the C2' position of the S
`moiety or the C3' position of the. S moiety.
`
`Still further, nucleotides in accordance with the
`practices of this invention include the nucleotides
`having the formula
`Sig
`
`- S - B
`P
`wherein P is the phosphcric acid moiety,, S the sugar
`moiety and B the base moiety.
`In these special nucleo(cid:173)
`tides, the P moiety is attached to the 3' and/or the 5'
`position of the S moiety when the nucleotide is deoxyribo(cid:173)
`nucleotide and at the 2', 3' and/or 5' position when
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Page 16 of 108
`
`

`
`........
`
`-17-
`
`the nucleotide is a ribonucleotide. The base B is
`either a purine or a pyrimidine and the B moiety is
`attached from the Nl or the N9 position to the 1'
`position of the sugar moiety when said B moiety is a.
`pyrimidine or a purine, respectively. The Sig chemical
`moiety is covalently attached to the phosphoric acid P
`..
`moiety via the chemical linkage
`OH
`
`- P - 0 - Sig
`II
`0
`said Sig, when attached to said P moiety being capable
`of signalling itself or making itself self-detecting or
`its presence known and desirably the nucleotide is
`capable of being incorporated into a double-stranded
`polynucleotide, such as DNA, RNA or DNA-RNA hybrid and
`when so incorporated therein is still 'self-detecting.
`
`5
`
`10
`
`15
`
`20
`
`It is pointed out that the special nucleotides in
`accordance with the practices of this invention des(cid:173)
`cribed or defined hereinabove by the general formula
`P - S - B - Sig, also include nucleotides wherein the
`Sig chemical moiety is covalently attached to the B
`25 moiety at the N6 or 6~amino group position when the B
`moiety is adenine or the N2 or 2-amino group position
`when the B moiety is guanine or the N4 or 4-amino group
`position when the B moiety is cytosine. The resulting
`nucleotides containing the Sig moiety attached thereto
`are capable of signalling themselves or making them(cid:173)
`selves self-detecting or their presence known and being
`detectable is a double-stranded or DNA, RNA or DNA-RNA
`hybrid.
`
`30
`
`! . /
`
`Page 17 of 108
`
`

`
`-18-
`
`By way of summary, as indicated hereinabove with
`respect to the make up of the various special nucleo(cid:173)
`tides in.accordance with this invention, the special
`nucleotides can be described as comprising a phosphoric
`acid moiety P, a sugar moiety s and a base moiety B, a
`pur.ine or pyrimidine, which combin<ltion of P-S-B is
`well known with respect to end defines nucleotides,
`both deoxyribonucleotides and ribonucleotides. The
`nucleotides are th~n modified in accordance with the
`practices of this invention by having covalently
`attaci!ed then<to, t·o the P moiety a·nd/or the s moiety
`and/or the B moiety, a chemical moiety Sig. The
`chemical moiety Sig so attached to the nucleotide P-5-B.
`i~ capable of rendering or making the res~lting nucleo-
`tide, now comprising P-S-B with the Sig moiety being
`attached to one or more of the other moieties, self(cid:173)
`detecting or signalling itself or capable of making its
`presence known per se, when incc·rporated into· a poly(cid:173)
`nucleotide, especially a double-stranded polynucleo-
`tide, such as a double-stranded DNA, a double-stranded
`RNA or a double-stranded DNA-RNA hybrid. The Sig
`moiety desirably should not interfere with the capa(cid:173)
`bility of the nucleotide to form a do0ble-stranded
`polynucleotide containing the special Sig-containing
`nucleotide in accordance with this invention and, when
`so incorporated therein, the Sig~containing nucleotide
`is capable of detection, localization or observation.
`
`The Sig moiety employed in the make up of the special
`nucleotides of this invention could comprise an enzyme
`or enzymatic material, such as alkaline phosphatase,
`glucose oxidase, horseradish peroxidase or ribonuclease.
`The Sig moiety could also co:•tain a fluorescing com(cid:173)
`ponent, such as fluoresc.ein or rhodamine or dansyl.
`desired, the Sig moiety could include a magnetic com(cid:173)
`ponent associated or attached thereto, such as a
`magnetic oxide or magnetic iron oxide, which would
`
`If
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Page 18 of 108
`
`

`
`·-
`
`-19-
`
`make the nuclectide cr polynucleotide containing such a
`magnetic-containing Sig moiety detectable by magnetic
`means. The Sig moiety might also include an electron
`dense component, such as ferritin, so as to be
`available by observation. The Sig moiety cculd also
`include a radioactive isotope component, such as
`radioactive cobalt, making the resulting nucleotide
`observable bx radiation detecting means. The Sig
`moiety could also include a hapten component or per se
`be c"-pable of complexing with an antibody specific
`thereto. Most usefully, the Sig moiety is a
`pcly&accharide or oligosaccharide or monosaccharide,
`which is capable of cowplexing with or being attached
`to a sugar or polysaccharide binding ~rotein, such as a
`lectin, e.g. Concanavilin A. The Sig component or
`moiety of the special nucleotides in accordance with
`this invention could also include a chemiluminescent·
`component.
`
`As· indicated in accordance with the ~racticei of this
`invention, the Sig component could comprise any
`chemical moiety which is attachable either directly or
`through a chemical linkage or linker arm to the
`qucleotide, such as to the base B component therein, or ~
`the sugar S component therein, or the phosphoric acid P
`component thereof.
`
`The Sig com~onent of the nucleotides in accordance with
`this invention and the nucleotides and pclynucleotides
`incorporating the nucleotides of this invention
`containing the Sig component are equivalent to and
`useful for the same purposes as the nucleotides
`described in the above-identified u.s. patent applica(cid:173)
`tion Serial No. 255,223. More specifically, the
`chemical.moiety A described in u.s. patent aFplication
`Serial No. 255,223 is functionally the equ~valent of
`the Sig component or chemical moiety of the special
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`/
`
`Page 19 of 108
`
`

`
`-20-
`
`nucleotides of this invention. Accordingly, the Sig
`component or chemical moiety of nucleotides of this
`invention can be directly covalently attached to the P,
`S or B moieties or attached thereto via a chemical
`linkage or linkage arm as described in U.S. patent
`application Ser. No. 255,223, as indicated by the
`dotted line connecting B and A of the nucleotides of
`U.S. Serial No. 255,223. The various linker arms or
`lin~ages identified in u.s. Ser. No. 255,223 are
`applicable to and useful in the preparation of the
`special nucleotides of this invention.
`
`A particularly important and bseful aspect of the
`special nucleotides of this invention is the use of
`such nucleotides in the. preparation of DNA or RNA
`probes. Such probes would contain a nucleotide .se(cid:173)
`quence substantially matching the DNA or RNA sequence
`of genetic material to be located and/or identified.
`The probe would contain one or more of the special
`nucleotides of this invention. A probe having a
`desired nucleotide sequence, such as a single-stranded
`polynucleotide, either DNA or RNA probe, would then be
`brought into contact with DNA or RNA genetic material
`to be identified. Upon the localization of the probe
`and the formation of a double-stranded polynucleotide
`containing the ptobe and thE: matching DNA or RNA
`material to be identified, the resulting formed do~ble­
`stranded DNA or RNA-containing material would then be
`observable and identified. A probe in accordance with
`this invention may contain substantially any number of
`nucleotide units, from about 5 nucleotides up to about
`500 or more, as may be required.
`It would appear that
`12 matching, preferably consecutive, nucleotide units
`would be sufficient to effect an identification of most
`of the DNA or RNA material to be investigated or
`identifieq, if the 12 nucleotide sequence of the probe
`matches a corresponding cooperative sequence in the DNA
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`. .
`..
`
`.~
`
`Page 20 of 108
`
`

`
`··~-
`
`-21-
`
`or RNA material being investigated or to be identified.
`As indicated, such probes may contain one or more of
`the special Sig-containing nucleotides in accordance
`with this invention, preferably at least about one
`special nucleotide per 5-10 of the nucleotides in the
`probe.
`
`5
`
`The disclosures of the above-identified PNAS article
`and•u.s. patent applications Serial No 255,223, filed
`10 April 17, 1981 and Serial No. 391,440, filed June 23,
`1982 and U.S. Patent 4,358,535 are here incorporated
`and made part of· this disclosure.
`
`15
`
`SUMMARY OF THE INVENTION
`
`20
`
`In accordance.w