`

`U.S. PATENT DOCUMENTS
`
`5,534,424 A
`5,547,839 A
`5,547,859 A
`5,602,000 A
`5,763,594 A
`5,770,367 A
`5,798,210 A
`5,808,045 A
`5,821,356 A
`5,849,542 A
`5,872,244 A
`5,885,775 A
`6,001,566 A
`6,008,379 A
`6,046,005 A
`6,074,823 A
`6,087,095 A
`6,136,543 A
`6,214,987 Bl
`6,218,118 Bl
`6,218,530 Bl
`6,232,465 Bl
`6,242,193 Bl
`6,255,475 Bl
`6,287,821 Bl
`6,309,836 Bl
`6,312,893 Bl
`6,380,378 Bl
`6,524,829 Bl
`6,613,508 Bl
`6,639,088 B2
`6,664,079 B2
`6,780,591 B2
`6,787,308 B2
`6,911,345 B2
`6,982,146 Bl
`7,037,687 B2
`7,056,666 B2
`7,057,026 B2
`7,057,031 B2
`7,074,597 B2
`7,078,499 B2
`7,105,300 B2
`7,414,116 B2
`7,427,673 B2
`7,459,275 B2
`2003/0008285 Al
`2003/0186256 Al
`2004/0014096 Al
`2004/0096825 Al
`2007/0166705 Al
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`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`WO
`WO
`WO
`WO
`WO
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`WO
`WO
`
`0992511
`1182267
`1291354
`0808320
`1337541
`1218391
`1790736
`WO 89/09282
`WO 90/13666
`WO 91/06678
`WO 92/10587
`WO 93/05183
`WO 93/21340
`WO 94/14972
`WO 96/07669
`WO 96/23807
`WO 96/27025
`
`4/2000
`2/2002
`3/2003
`4/2003
`3/2007
`4/2007
`5/2007
`10/1989
`11/1990
`* 5/1991
`6/1992
`3/1993
`10/1993
`7 /1994
`3/1996
`8/1996
`9/1996
`
`US 7,566,537 B2
`Page 2
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`WO
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`WO
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`WO
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`WO
`WO
`WO
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`WO 98/30720
`WO 99/05315
`WO 99/57321
`WO 00/02895
`WO 00/06770
`WO 00/15844
`WO 00/18956
`WO 00/21974
`WO 00/50642
`WO 00/53805
`WO 00/53812
`WO 00/70073
`WO 01/16375
`WO 01/23610
`WO 01/25247
`WO 01/32930
`WO 01/57248
`WO 01/57249
`WO 01/92284
`WO 02/02813
`WO 02/22833
`WO 02/29003
`WO 02/072892
`WO 02/079519
`WO 02/088381
`WO 02/088382
`WO 03/002767
`WO 03/020968
`WO 03/048178
`WO 03/048387
`WO 03/085135
`WO 2004/007773
`WO 2004/018493
`WO 2004/018497
`WO 2005/084367
`
`7 /1998
`2/1999
`11/1999
`1/2000
`2/2000
`3/2000
`4/2000
`4/2000
`8/2000
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`3/2001
`4/2001
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`11/2002
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`3/2003
`6/2003
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`10/2003
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`3/2004
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`3'-tags, Gene, 148: 1-6 (1994).
`Metzker et al., Termination of DNA synthesis by novel 3'-modified(cid:173)
`deoxyribonucleoside 5'-triphosphates, Nucleic Acids Research, 22:
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`Hovinen et al., Synthesis of 3'-O-(m-Aminoalkoxymethyl)
`thymidine 5'-Triphosphates, Terminators of DNA Synthesis that
`Enable 3'-Labelling, J. Chem. Soc. Perkin Trans., 1: 211-217 (1994).
`Stratagene Catalog, p. 39 ( 1988).
`Kraevskii et al., Substrate Inhibitors of DNA Biosynthesis, Trans(cid:173)
`lated from Molekulyamaya Biologiya [Mo!. Bio. (Mosk.)]21:33-38
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`WO 2004/018497, Intl. Search Report, PCT.
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`

`

`US 7,566,537 B2
`Page 3
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`Ikeda et al., A Non-radioactive DNA Sequencing Method Using
`Biothinylated Dideoxynucleoside Triphosphates and 1Hth DNA
`Polymerase, 2:225-227 (1995).
`Welch et al., Syntheses ofNucleosides Designed for Combinatorial
`DNA Sequencing, Chemistry, European Jouranl, 5:951-960 (1999).
`Canard et al., Catalytic editing properties ofDNA polymerases, Proc.
`Natl. Acad. Sci., 92:10859-10863 (1995).
`Olejnik et al., Photocleavable biotin derivatives: A versatile approach
`for the isolation of biomolecules, Proc. Natl. Acad. Sci., 92:7590-
`7594 (1995).
`Wada et al., 2-(Azidomethyl)benzoyl as a new protecting group in
`nucleosides, Tetrahedron Letters, 42:1069-1072 (2001).
`Li et al., A photocleavable fluorescent nucleotide ofDNA sequencing
`and analysis, Proc. Natl. Acad. Sci., 100:414-419 (2003).
`Sarfati et al., Synthesis of fluorescent derivatives of 3'-O-(6-
`aminohexanoyl)-pyrimidine nucleosides 5'-triphosphates that act as
`DNA polymerase substrates reversibly tagged at C-3', JCS Perkin
`Trans, 1163-1171 (1995).
`Burgess et al., An Approach to Photolabile, Fluorescent Protecting
`Groups, J. Org. Chem., 62:5165-5168 (1997).
`Rasolonjatovo et al., 6-N-
`(N-Methylanthranylamido)-4-Oxo(cid:173)
`Hexanoic Acid: A New Fluorescent Protecting Group Applicable to
`a New DNA Sequencing Method, Nucleosides & Nucleotides,
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`Marquez et al., Selective Fluorescence Quenching of 2,3-
`Diazabicyclo[2.2.2]oct-2-ene by Nucteotides, Organic Letters,
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`oligodeoxyribonucleotides on the fluorescent properties of conju(cid:173)
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`Kvam et al., Characterization of singlet oxygen-induced guanine
`residue damage after photochemical treatment of free nucleosides
`and DNA, Biochimica et Biophysica Acta., 1217:9-15 ( 1994).
`Crespo-Hernandez et al., Part 1. Photochemical and Photophysical
`Studies of Guanine Derivatives: Intermediates Contributing to its
`Photodestruction Mechanism in Aqueous Solution and the Participa(cid:173)
`tion of the Electron Adduct, Photochemistry and Photobiology,
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`sorbing Flourescent Dyes, Bioconjugate Chem., 14: 195-204 (2003).
`Torimura et al., Fluorescence-Quenching Phenomenon by
`Photoinduced Electron Transfer between a Fluorescent Dye and
`Nucleotide Base, Analytical Sciences, 17:155-160 (2001).
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`labelled oligonucleotides and their application in DNA sequencing,
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`* cited by examiner
`
`

`

`U.S. Patent
`
`Jul. 28, 2009
`
`Sheet 1 of 6
`
`US 7,566,537 B2
`
`Uridine C5-linker
`
`Cytidine CS-linker
`
`Linker,Label
`
`Linker, Label
`
`0
`
`N
`
`NH2
`
`XO
`
`R1
`
`R2
`
`N~N
`
`N7 Deazaadenosine C7-linker
`
`0
`
`N
`0
`XO~
`R2 NyN-H
`R1
`NH2
`N7 Deazaguanosine C7 -linker
`
`Adenosine N6-linker
`
`Cytidine N4-linker
`
`where R1 and R2 , which may be the
`same or different, are each selected
`from H, OH, or any group which can be
`transformed into an OH. Suitable groups
`for R1 and R2 are described in Figure 3
`
`t='i_no
`o
`xo~YT
`R2 NlN_H
`R1
`HN,
`Linker-Label
`
`X = H, phosphate, diphosphate or triphosphate
`
`Guanosine N2-linker
`
`Fig. 1
`
`

`

`U.S. Patent
`
`Jul. 28, 2009
`
`Sheet 2 of 6
`
`US 7,566,537 B2
`
`Acid Labile Linkers
`
`-1{0
`0~
`Dye ~'-~~o~ fo
`0
`OMe
`NH
`2: Dialkoxybenzyl linker ~
`
`base
`
`Disulfide Linker
`
`Dye)r-HN
`o
`o
`1-.......,.,s,s
`~
`NH
`~ base
`
`t
`
`-O;)o~
`-11..0
`N~O
`Dye N-.___~
`0
`'!(\/\,,.o
`--
`7 ~
`H
`0
`..--
`
`0
`
`3: Siebe, Unlm
`
`4: Indole Linker
`
`-11..0
`Dye N'-H
`H N'(\/"vo
`0
`
`5: tButyl Sieber Linker
`
`NH
`
`~
`
`base
`
`Fig. 2
`
`

`

`U.S. Patent
`
`Jul. 28, 2009
`
`Sheet 3 of 6
`
`US 7,566,537 B2
`
`Label =Cleavable linker-"''""'""""Base
`
`t-!-B
`
`Cleavable linkers may include:
`
`R,~ox
`R1
`
`where R 1 and R2 , which may be the same or
`different, are each selected from H, OH, or
`any group which can be transformed into an
`OH, including a carbonyl
`
`1:ti-s·L
`0 B¾
`Lt]'-~
`~o~ ------o
`'o
`.)<_Rs
`_,,kRs
`~ R.i 9
`R4
`Ru
`~
`
`R 1 and R2 groups may include
`
`'o
`,)<Rs
`R.i ~
`~
`
`'o
`'o
`,)<Rs
`\
`R6 Ri N3
`
`R3 represents one or more
`substituents independently
`selected from alkyl, alkoxy,
`amino or halogen
`
`Alternatively, cleavable linkers may
`be constructed from any labile
`functionality used on the 3'-block
`
`'o
`_)::Rs
`Rj
`F
`
`'o
`~I
`
`'o
`'o
`ARs
`aAo~ R.i 0~
`
`'o
`oJ-..o-~
`
`'o
`'o
`'o
`O.J-_N-~ oAs--~ o.J-.~
`
`where ~ is H or alkyl, R5 is H or
`alkyl and~ is alkyl, cycloalkyl,
`alkenyl, cycloalkenyl or benzyl
`
`and Xis H, phosphate, diphosphate or triphosphate
`
`Fig. 3
`
`

`

`Klenow exo- TMR
`dUTP
`
`pH7.5
`
`New disulfide cleavable linker (1)
`
`0 0.5 1 3 5
`
`+DTT
`0 0.5 1 3 5 min.
`
`Incorporation Cleavage
`
`AG
`
`AG
`
`50mM Tris-HCI pH7.5, lOmM NaCl, O.lmM EDTA
`5mM MgC12, 2uM dNTP-fluor, lOOnM SHP 5T hairpin AG oligo,
`Klenow exo- (Amersham-Joyce) I Ounits.
`
`~
`00
`•
`~
`~
`~
`
`~ = ~
`
`2' :-'
`
`N
`~CIO
`N
`0
`0
`1,0
`
`('D
`('D
`
`.i;...
`
`rJJ =(cid:173)
`.....
`0 ....
`
`O'I
`
`t = 0, 30s, 1,3, 5
`
`Fig. 4
`
`d r.,;_
`-....l
`tit
`0--,
`0--,
`tit w
`-....l = N
`
`

`

`New Acid cleavable Sieber linker (3)
`
`Klenow exo- TMR
`dUTP
`
`pH7.5
`
`(cid:141)
`
`0 1 3 5 10 min.
`
`AG
`
`50mM Tris-HCl pH7.5, lOmM NaCl, 2mM DTT, O.lmM EDTA
`SmM MgC12, 2uM dNTP-fluor, lOOnM SHP 5T hairpin AG oligo,
`Klenow exo- (Amersham-Joyce) 1 Ounits.
`
`~
`00
`•
`~
`~
`~
`
`~ = ~
`
`2' :-'
`
`N
`~CIO
`N
`0
`0
`1,0
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`Ul
`0 ....
`
`O'I
`
`t=O, 1, 3, 5, 10
`
`Fig. 5
`
`d r.,;_
`-....l
`tit
`0--,
`0--,
`tit w
`-....l = N
`
`

`

`New Acid cleavable lndole linker (4)
`
`Klenow exo- TMR
`dUTP
`
`pH7.5
`
`0 l 3 5 min
`
`AG
`
`50mM Tris-HCI pH7.5, lOmM NaCl, 2mM DTT, O. lmM EDTA
`5mM MgC12, 2uM dNTP-fluor, 1 OOnM SHP ST hairpin AG oligo,
`Klenow exo- (Amersham-Joyce) 1 Ounits.
`
`~
`00
`•
`~
`~
`~
`
`~ = ~
`
`2' :-'
`
`N
`~CIO
`N
`0
`0
`1,0
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`O'I
`0 ....
`
`O'I
`
`t=0,1,3,5
`
`Fig. 6
`
`d r.,;_
`-....l
`tit
`0--,
`0--,
`tit w
`-....l = N
`
`

`

`1
`LABELLED NUCLEOTIDES
`
`RELATED APPLICATIONS
`
`US 7,566,537 B2
`
`2
`SUMMARY OF THE INVENTION
`
`In the present invention, a nucleoside or nucleotide mol(cid:173)
`ecule is linked to a detectable label via a cleavable linker
`5 group attached to the base, rendering the molecule useful in
`techniques using Labelled nucleosides or nucleotides, e.g.,
`sequencing reactions, polynucleotide synthesis, nucleic acid
`amplification, nucleic acid hybridization assays, single nucle(cid:173)
`otide polymorphism studies, and other techniques using
`10 enzymes such as polymerases, reverse transcriptases, termi(cid:173)
`nal transferases, or other DNA modifying enzymes. The
`invention is especially useful in techniques that use Labelled
`dNTPs, such as nick translation, random primer labeling,
`end-labeling ( e.g., with terminal deoxynucleotidyltrans-
`ferase ), reverse transcription, or nucleic acid amplification.
`The molecules of the present invention are in contrast to the
`prior art, where the label is attached to the ribose or deoxyri(cid:173)
`bose sugar, or where the label is attached via a non-cleavable
`linker.
`According to a first aspect of the invention, a nucleotide or
`20 nucleoside molecule, or an analog thereof, has a base that is
`linked to a detectable label via a cleavable linker.
`The invention features a nucleotide or nucleoside mol(cid:173)
`ecule, having a base that is linked to a detectable label via a
`cleavable linker. The base can be a purine, or a pyrimidine.
`The base can be a deazapurine. The molecule can have a
`ribose or deoxyribose sugar moiety. The ribose or deoxyri(cid:173)
`bose sugar can include a protecting group attached via the 2'
`or 3' oxygen atom. The protecting group can be removed to
`expose a 3'-OH. The molecule can be a deoxyribonucleotide
`triphosphate. The detectable label can be a fluorophore. The
`linker can be an acid labile linker, a photo labile linker, or can
`contain a disulphide linkage.
`The invention also features a method oflabeling a nucleic
`acid molecule, where the method includes incorporating into
`the nucleic acid molecule a nucleotide or nucleoside mol(cid:173)
`ecule, where the nucleotide or nucleoside molecule has a base
`that is linked to a detectable label via a cleavable linker. The
`incorporating step can be accomplished via a terminal trans(cid:173)
`ferase, a polymerase or a reverse transcriptase. The base can
`be a purine, or a pyrimidine. The base can be a deazapurine.
`The nucleotide or nucleoside molecule can have a ribose or
`deoxyribose sugar moiety. The ribose or deoxyribose sugar
`can include a protecting group attached via the 2' or 3' oxygen
`atom. The protecting group can be removed to expose a 3'-OH
`group. The molecule can be a deoxyribonucleotide triphos-
`phate. The detectable label can be a fluorophore. The linker
`can be an acid labile linker, a photolabile linker, or can contain
`a disulphide linkage. The detectable label and/or the cleav-
`able linker can be of a size sufficient to prevent the incorpo(cid:173)
`ration of a second nucleotide or nucleoside into the nucleic
`acid molecule.
`In another aspect, the invention features a method for deter-
`mining the sequence of a target single-stranded polynucle(cid:173)
`otide, where the method includes monitoring the sequential
`incorporation of complementary nucleotides, where the
`nucleotides each have a base that is linked to a detectable label
`55 via a cleavable linker, and where the identity of each nucle(cid:173)
`otide incorporated is determined by detection of the label
`linked to the base, and subsequent removal of the label.
`The invention also features a method for determining the
`sequence of a target single-stranded polynucleotide, where
`the method includes: (a) providing nucleotides, where the
`nucleotides have a base that is linked to a detectable label via
`a cleavable linker, and where the detectable label linked to
`each type of nucleotide can be distinguished upon detection
`from the detectable label used for other types of nucleotides;
`(b) incorporating a nucleotide into the complement of the
`target single stranded polynucleotide; ( c )detecting the label
`of the nucleotide of (b ), thereby determining the type of
`nucleotide incorporated; ( d) removing the label of the nucle-
`
`This application is a Divisional Application of U.S. appli(cid:173)
`cation Ser. No. 10/227,131, now U.S. Pat. No. 7,057,026 filed
`Aug. 23, 2002 which in turn claims benefit of United King(cid:173)
`dom Application No. GB0129012.1, filed Dec. 4, 2001. The
`entire teachings of the above applications are incorporated
`herein by reference.
`
`FIELD OF THE INVENTION
`
`This invention relates to labelled nucleotides. In particular,
`this invention discloses nucleotides having a removable label 15
`and their use in polynucleotide sequencing methods.
`
`BACKGROUND
`
`Advances in the study of molecules have been led, in part,
`by improvement in technologies used to characterise the mol(cid:173)
`ecules or their biological reactions. In particular, the study of
`the nucleic acids DNA and RNA has benefited from develop(cid:173)
`ing technologies used for sequence analysis and the study of
`hybridisation events.
`An example of the technologies that have improved the 25
`study of nucleic acids, is the development of fabricated arrays
`of immobilised nucleic acids. These arrays consist typically
`of a high-density matrix of polynucleotides immobilised onto
`a solid support material. See, e.g., Fodor et al., Trends Bio(cid:173)
`tech. 12: 19-26, 1994, which describes ways ofassembling the 30
`nucleic acids using a chemically sensitized glass surface pro(cid:173)
`tected by a mask, but exposed at defined areas to allow attach(cid:173)
`ment of suitably modified nucleotide phosphoramidites. Fab(cid:173)
`ricated arrays can also be manufactured by the technique of
`"spotting" known polynucleotides onto a solid support at 35
`predetermined positions (e.g., Stimpson et al., Proc. Natl.
`Acad. Sci. USA 92:6379-6383, 1995).
`A further development in array technology is the attach(cid:173)
`ment of the polynucleotides to the solid support material to
`form single molecule arrays. Arrays of this type are disclosed
`in International Patent App. WO 00/06770. The advantage of
`these arrays is that reactions can be monitored at the single
`molecule level and information on large numbers of single
`molecules can be collated from a single reaction.
`For DNA arrays to be useful, the sequences of the mol(cid:173)
`ecules must be determined. U.S. Pat. No. 5,302,509 discloses 45
`a method to sequence polynucleotides immobilised on a solid
`support. The method relies on the incorporation of3'-blocked
`bases A, G, C and T having a different fluorescent label to the
`immobilised polynucleotide, in the presence of DNA poly(cid:173)
`merase. The polymerase incorporates a base complementary 50
`to the target polynucleotide, but is prevented from further
`addition by the 3'-blocking group. The label of the incorpo(cid:173)
`rated base can then be determined and the blocking group
`removed by chemical cleavage to allow further polymerisa(cid:173)
`tion to occur.
`Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) describes
`the synthesis of nucleotide triphosphates modified with a
`3'-O-blocking group that is photolabile and fluorescent. The
`modified nucleotides are intended for use in DNA sequencing
`experiments. However, these nucleotides proved to be diffi(cid:173)
`cult to incorporate onto an existing polynucleotide, due to an 60
`inability to fit into the polymerase enzyme active site.
`Zhu et al. (Cytometry 28:206-211, 1997) also discloses the
`use of fluorescent labels attached to a nucleotide via the base
`group. The labelled nucleotides are intended for use in fluo(cid:173)
`rescence in situ hybridisation (FISH) experiments, where a 65
`series of incorporated labelled nucleotides is required to pro(cid:173)
`duce a fluorescent "bar code".
`
`40
`
`

`

`US 7,566,537 B2
`
`10
`
`3
`otide of (b ); and ( e) optionally repeating steps (b )-( d) one or
`more times; thereby determining the sequence of a target
`single-stranded polynucleotide.
`In the methods described herein, each of the nucleotides
`can be brought into contact with the target sequentially, with 5
`removal of non-incorporated nucleotides prior to addition of
`the next nucleotide, where detection and removal of the label
`is carried out either after addition of each nucleotide, or after
`addition of all four nucleotides.
`In the methods, all of the nucleotides can be brought into
`contact with the target simultaneously, i.e., a composition
`comprising all of the different nucleotides is brought into
`contact with the target, and non-incorporated nucleotides are
`removed prior to detection and subsequent to removal of the 15
`label(s).
`The methods can comprise a first step and a second step,
`where in the first step, a first composition comprising two of
`the four nucleotides is brought into contact with the target,
`and non-incorporated nucleotides are removed prior to detec- 20
`tion and subsequent to removal of the label, and where in the
`second step, a second composition comprising the two nucle(cid:173)
`otides not included in the first composition is brought into
`contact with the target, and non-incorporated nucleotides are
`removed prior to detection and subsequent to removal of the 25
`label, and where the first steps and the second step can be
`optionally repeated one or more times.
`The methods described herein can also comprise a first step
`and a second step, where in the first step, a composition 30
`comprising one of the four nucleotides is brought into contact
`with the target, and non-incorporated nucleotides are
`removed prior to detection and subsequent to removal of the
`label, and where in the second step, a second composition.
`comprising the three nucleotides not included in the first 35
`composition is brought into contact with the target, and non(cid:173)
`incorporated nucleotides are removed prior to detection and
`subsequent to removal of the label, and where the first steps
`and the second step can be optionally repeated one or more
`times.
`The methods described herein can also comprise a first step
`and a second step, where in the first step, a first composition
`comprising three of the four nucleotides is brought into con(cid:173)
`tact with the target, and non-incorporated nucleotides are
`removed prior to detection and subsequent to removal of the 45
`label, and where in the second step, a composition comprising
`the nucleotide not included in the first composition is brought
`into contact with the target, and non-incorporated nucleotides
`are removed prior to detection and subsequent to removal of
`the label, and where the first steps and the second step can be 50
`optionally repeated one or more times.
`In a further aspect, the invention features a kit, where the kit
`includes: (a) individual the nucleotides, where each nucle(cid:173)
`otide has a base that is linked to a detectable label via a
`cleavable linker, and where the detectable label linked to each
`nucleotide can be distinguished upon detection from the
`detectable label used for other three nucleotides; and (b)
`packaging materials therefor. The kit can further include an
`enzyme and buffers appropriate for the action of the enzyme.
`The nucleotides/nucleosides are suitable for use in many
`different DNA-based methodologies, including DNA synthe(cid:173)
`sis and DNA sequencing protocols.
`According to another aspect of the invention, a method for
`determining the sequence of a target polynucleotide com(cid:173)
`prises monitoring the sequential incorporation of comple- 65
`mentary nucleotides, wherein the nucleotides comprise a
`detectable label linked to the base portion of the nucleotide
`
`4
`via a cleavable linker, incorporation is detected by monitoring
`the label, and the label is removed to permit further nucleotide
`incorporation to occur.
`
`DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shows examplary nucleotide structures useful in the
`invention. For each structure, X can be H, phosphate, diphos(cid:173)
`phate or triphosphate. R 1 and R2 can be the same or different,
`and can be selected from H, OH, or any group which can be
`transformed into an OH, including, but not limited to, a car(cid:173)
`bonyl. Some suitable functional groups for R 1 and R2 include
`the structures shown in FIG. 3.
`FIG. 2 shows structures of linkers useful in the invention,
`including (1) disulfide linkers and acid labile linkers, (2)
`dialkoxybenzyl linkers, (3) Sieber linkers, (4) indole linkers
`and (5) t-butyl Sieber linkers.
`FIG. 3 shows some functional molecules useful in the
`invention, including some cleavable linkers and some suit(cid:173)
`able hydroxyl protecting groups. In these structures, R 1 and
`R2 may be the same of different, and can be H, OH, or any
`group which can be transformed into an OH group, including
`a carbonyl. R3 represents one or more substituents indepen(cid:173)
`dently selected from alkyl, alkoxyl, amino or halogen groups.
`Alternatively, cleavable linkers may be constructed from any
`labile functionality used on the 3'-block. R4 and R5 can be H
`or alkyl, and R 6 can be alkyl, cycloalkyl, alkenyl, cycloalk(cid:173)
`enyl or benzyl. X can be H, phosphate, diphosphate or triph-
`osphate.
`FIG. 4 shows a denaturing gel showing the incorporation of
`the triphosphate of Example 1 using Kienow polymerase.
`FIG. 5 shows a denaturing gel showing the incorporation of
`the triphosphate of Example 3 using Kienow polymerase.
`FIG. 6 shows a denaturing gel showing the incorporation of
`the triphosphate of Example 4 using Kienow polymerase.
`
`DETAILED DESCRIPTION
`
`The present invention relates to nucleotides and nucleo(cid:173)
`sides that are modified by attachment of a label via a cleavable
`linker, thereby rendering the molecule useful in techniques
`40 where the labelled molecule is to interact with an enzyme,
`such as sequencing reactions, polynucleotide synthesis,
`nucleic acid amplification, nucleic acid hybridization assays,
`single nucleotide polymorphism studies, techniques using
`enzymes such as polymerase, reverse transcriptase, terminal
`transferase, techniques that use Labelled dNTPs ( e.g., nick
`translation, random primer labeling, end-labeling (e.g., with
`terminal deoxynucleotidyltransferase ), reverse transcription,
`or nucleic acid amplification).
`As is known in the art, a "nucleotide" consists of a nitrog(cid:173)
`enous base, a sugar, and one or more phosphate groups. In
`RNA, the sugar is a ribose, and in DNA is a deoxyribose, i.e.,
`a sugar lacking a hydroxyl group that is present in ribose. The
`nitrogenous base is a derivative of purine or pyrimidine. The
`purines are adenosine (A) and guanidine (G), and the pyrim(cid:173)
`idines are cytidine (C) and thymidine (T) ( or in the context of
`55 RNA, uracil (U)). The C-1 atom of deoxyribose is bonded to
`N-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a
`phosphate ester of a nucleoside, with esterification occurring
`on the hydroxyl group attached to C-5 of the sugar. Nucle(cid:173)
`otides are usually mono, di- or triphosphates.
`A "nucleoside" is structurally similar to a nucleotide, but
`60 are missing the phosphate moieties. An example of a nucleo(cid:173)
`side analog would be one in which the label is linked to the
`base and there is no phosphate group attached to the sugar
`molecule.
`Although the base is usually referred to as a purine or
`pyrimidine, the skilled person will appreciate that derivatives
`and analogs are available which do not alter the capability of
`the nucleotide or nucleoside to undergo Watson-Crick base
`
`

`

`6
`erence to DNA, although the description will also be
`applicable to RNA, PNA, and other nucleic acids, unless
`otherwise indicated.
`The modified nucleotides of the invention use a cleavable
`linker to attach the label to the nucleotide. The use of a
`cleavable linker ensures that the label can, if required, be
`removed after detection, avoiding any interfering signal with
`any labelled nucleotide incorporated subsequently.
`Cleavable linkers are known in the art, and conventional
`chemistry can be applied to attach a linker to a nucleotide base
`and a label. The linker can be cleaved by any suitable method,
`including exposure to acids, bases, nucleophiles, electro(cid:173)
`philes, radicals, metals, reducing or oxidising agents, light,
`temperature, enzymes etc. Suitable linkers can be adapted
`from standard chemical blocking groups, as disclosed in
`Greene & Wuts, Protective Groups in Organic Synthesis,
`John Wiley & Sons. Further suitable cleavable linkers used in
`solid-phase synthesis are disclosed in Guillier et al. (Chem.
`Rev. 100:2092-2157, 2000).
`The use of the term "cleavable linker" is not meant to imply
`that the whole linker is required to be removed from the
`nucleotide base. The cleavage site can be located at a position
`on the linker that ensures that part of the linker remains
`attached to the nucleotide base after cleavage.
`The linker can be attached at any position on the nucleotide
`base provided that Watson-Crick base pairing can still be
`carried out. In the context of purine bases, it is preferred if the
`linker is attached via the 7 position of the purine or the
`preferred deazapurine analogue, via an 8-modified purine, via
`an N-6 modified adenosine or an N-2 modified guanine. For
`pyrimidines, attachment is preferably via the 5 position on
`cytidine, thymidine or uracil and the N-4 position on cytosine.
`Suitable nucleotide structures are shown in FIG. 1. For each
`structure in FIG. 1, X can be H, phosphate, diphosphate or
`triphosphate. R 1 and R2 can be the same or different, and can
`be selected from H, OH, or any group which can be trans(cid:173)
`formed into an OH, including, but not limited to, a carbonyl.
`Some suitable functional groups for R 1 and R2 include the
`structures shown in FIG. 3.
`Suitable linkers are shown in FIG. 2 and include, but are not
`limited to, disulfide linkers (1 ), acid labile linkers (2, 3, 4 and
`5; including dialkoxybenzyl linkers (e.g., 2), Sieber linkers
`(e.g., 3), indole linkers (e.g., 4), t-butyl Sieber linkers (e.g.,
`5)), electrophilically cleavable
`linkers, nucleophilically
`cleavable linkers, photocleavable linkers, cleavage under
`reductive conditions, oxidative conditions, cleavage via use
`of safety-catch linkers, and cleavage by elimination mecha(cid:173)
`msms.
`
`A. Electrophilically Cleaved Linkers.
`Electrophilically cleaved linkers are typically cleaved by
`protons and include cleavages sensitive to acids. Suitable
`linkers include the modified benzylic systems such as trityl,
`p-alkoxybenzyl esters and p-alkoxybenzyl amides. Other
`suitable linkers include tert-butyloxycarbonyl (Boe) groups
`and the acetal system (e.g., as is shown in FIG. 3 as 0----C
`(R4 )(R5)---0-R6 .
`The use ofthiophilic metals, such as nickel, silver or mer(cid:173)
`cury, in the cleavage ofthioacetal or other sulphur-containing
`protecting groups can also be considered for the preparation
`of suitable linker molecules.
`
`5
`pairing. "Derivative" or "analog" means a compound or mol(cid:173)
`ecule whose core structure is the same as, or closely
`resembles that of, a parent compound, but which has a chemi-
`cal or physical modification, such