(12) United States Patent
`Burgess et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006340750Bl
`US 6,340,750 Bl
`Jan.22,2002
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) THROUGH BOND ENERGY TRANSFER IN
`FLUORESCENT DYES FOR LABELLING
`BIOLOGICAL MOLECULES
`
`(75)
`
`Inventors: Kevin Burgess, Bryan; Richard Gibbs,
`Houston, both of TX (US)
`
`(73) Assignee: The Texas A&M University System,
`College Station, TX (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/460,718
`
`(22) Filed:
`
`Dec. 14, 1999
`
`Related U.S. Application Data
`(60) Provisional application No. 60/112,711, filed on Dec. 18,
`1998.
`
`(51)
`
`Int. Cl? ........................ C07H 19/04; C07H 21!00;
`C09K 11/06; GOlN 21/01; GOlN 21/64
`
`(52) U.S. Cl. .............. 536/26.6; 536/25.32; 252/301.16;
`422/82.06; 422/82.07
`
`(58) Field of Search ........................... 435/6, 91.1, 183,
`435/283.1, 287.2; 436/94; 536/23.1, 24.3,
`24.33, 25.32, 25.3, 26.6, 26.41; 252/301.16;
`422/82.06, 82.07
`
`(56)
`
`References Cited
`
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`Nucleic Acids Res. 21, 5727-5735, Dec. 1993.*
`Kollmannsberger et al., Electrogenerated chemilumines(cid:173)
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`functionalized
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`Geohegan, Improved method for converting an unmodified
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`Dodd, C. R. Connell, C. Heiner, S. B. Kent, and L. E. Hood,
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`Large-Scale and Automated DNA Sequence Determination,
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`Large-Scale DNA Sequencing, T. Hunkapiller, R. J. Kaiser,
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`The Future of DNA Sequencing, L. M. Smith, Science,
`1993, 262, 530-2.
`A system for rapid DNA sequencing with fluorescent chain(cid:173)
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`R. J. Dam, F. W. Hobbs, C. W. Robertson, R. J. Zagursky,A
`J. Cocuzza, M.A. Jensen, and K. Baumeister, Science, 1987,
`238, 336.
`Energy Transfer Primers: A New Fluorescence Labeling
`Paradigm for DNA Sequencing and Analysis, J. Ju, A N.
`Glazer, and R. A Mathies, Nature Med., 1996, 2, 246-9.
`
`(List continued on next page.)
`
`Primary Examiner-Ethan Whisenant
`Assistant Examiner-Frank Lu
`(74) Attorney, Agent, or Firm-Baker Botts, L.L.P.
`
`(57)
`
`ABSTRACT
`
`Fluorescent energy transfer cassettes that allow through
`bond energy transfer and have a succinimidyl ester func(cid:173)
`tionality suitable for affecting them to biomolecules or
`provided and are applied to high throughput DNA sequenc(cid:173)
`ing.
`
`8 Claims, 13 Drawing Sheets
`
`a Through space FET from a donor dye
`D to an acceptor dye A;
`b through bond FET.
`
`a through space FET
`
`b through bond FET
`
`~v
`
`D
`
`1
`
`TFS1013
`
`

`

`US 6,340,750 Bl
`Page 2
`
`01HER PUBLICATIONS
`
`Fluorescence energy transfer dye-labeled primers for DNA
`sequencing and analysis, 1. 1u, C. Ruan, C. W. Fuller, A. N.
`Glazer, and R. A Mathies, Proc. Natl. Acad. Sci. USA, 1995,
`92, 4347-51.
`Ultra-High-Speed DNA Sequencing Using Capillary Elec(cid:173)
`trophoresis Chips, A T. Woolley, and R. A Mathies, Anal.
`Chern., 1995, 67, 3676-80.
`Rapid Sizing of Short Tandem Repeat Alleles Using Capil(cid:173)
`lary Array Electrophoresis and Energy-Transfer Fluorescent
`Primers, Y. Wang, 1. 1u, B. A. Carpenter, 1. M. Atherton, G.
`F. Sensabaugh, and R. A Mathies, Anal. Chern., 1995, 67,
`1197-203.
`Design and Synthesis of Fluorescence Energy Transfer
`Dye-Labeled Primers and Their Application for DNA
`Sequencing and Analysis, 1. 1u, I. Kheterpal, 1. R. Scherer,
`C. Ruan, C. W. Fuller, A N. Glazer, and R. A Mathies,
`Analytical Biochem., 1995, 231, 131-40.
`Energy
`Transfer
`Primers
`with
`or
`5-
`6-Carboxyrhodamine-6G as Acceptor Chromophores,
`S.-C. Hung, 1. 1u, R. A Mathies, and A N. Glazer, Ana(cid:173)
`lytical Biochem., 1996, 238, 165-70.
`Continuous, On-line DNA Sequencing Using Oligodeoxy(cid:173)
`nucleotide Primers With Multiple Fluorophores, 1. A Brum(cid:173)
`baugh, L. R. Middendorf, D. L. Grone, and 1. L. Ruth, Proc.
`Natl. Acad. Sci., 1988, 85, 5610-4.
`A Single Residue in DNA Polymerases of the Escherichia
`coli DNA Polymerase I Family is Critical for Distinguishing
`between Deoxy- and Dideoxyribonucleotides, S. Tabor, and
`C.C. Richardson, Proc. Natl. Acad. Sci. USA, 1995, 92,
`6339-6343.
`Cassette Labeling for Facile Construction of Energy Trans(cid:173)
`fer Fluorescent Primers, 1. 1u, A. N. Glazer, and R. A.
`Mathies, Nucleic Acids Res., 1996, 24, 1144-8.
`Electrophoretically Uniform Fluorescent Dyes for Auto(cid:173)
`mated DNA Sequencing, M. L. Metzker, 1. Lu, and R. A
`Gibbs, Science, 1996, 271, 1420-2.
`Comparison of Fluorescence Energy Transfer primers with
`Different Donor-Acceptor Dye Combinations, S. Hung,
`R.A Mathies, and A N. Glazer, Analytical Biochemistry,
`1998, 255, 32-8.
`A Convenient Synthesis of Acetylenes: Catalytic Substitu(cid:173)
`tions of Acetylenic Hydrogen with Bromoalkenes, Iodoare(cid:173)
`nes, and Bromopyridines, K. Sonogashira, Y. Tohda, and N.
`Hagihara, Tetrahedron Lett., 1975, 4467-70.
`Stepwise Synthesis of Substituted Oligo(phenylenevi(cid:173)
`nylene) via an Orthogonal Approach, T. Maddux, W. Li, and
`L. Yu, J. Am. Chern. Soc., 1997, 119, 844-5.
`Synthesis of Sequence Specific Phenylacetylene Oligomers
`on an Insoluble Solid Support, 1. K. Young, 1. C. Nelson, and
`1. S. Moore, 1. Am. Chern. Soc., 1994, 116, 10841-2.
`Iterative Divergent/Convergent Approach to Linear Conju(cid:173)
`gated Oligomers by Successive Doubling of the Molecular
`Length: A Rapid Route to a 128A-Long Potential Molecular
`Wire, 1. S. Schumm, D. L. Pearson, and 1. M. Tour,Angew.
`Chern., Int. Ed. Engl., 1994, 33, 1360-3.
`Soluble poly(1,4-phenyleneethynylene)s, R. Giesa, and R.
`C. Schulz, Makromol Chern., 1990, 191, 857-67.
`Luminescent Alkoxy-Functionalized Polyphenylenes, 1. L.
`Reddinger, and 1. R. Reynolds, Abstr. Papers Am Chern.
`Soc., 1996, 211, 530-1.
`
`Fluorescence Studies of Poly(p-phenyleneethynylene )s:
`The Effect of Andiracene Substitution, T. M. Swager, C. 1.
`Gil, and M.S. Wrighton,J. Phys. Chern., 1995,99,4886-93.
`
`Efficient Solid-State Photoluminescence in New Poly(2,
`5--dialkoxy-p-phenyleneethynylene)s, C. Weder, and M.S.
`Wrighton, Marcomolecules, 1996, 29, 5157-65.
`
`Energy Transfer in Dendritic Macromolecules: Molecular
`Size Effects and the Role of an Energy Gradient, C. Deva(cid:173)
`doss, P. Bharathi, and 1. S. Moore, J. Am. Chern. Soc., 1996,
`118, 9635-44.
`
`Phenylacetylene Dendrimers by the Divergent Convergent,
`and Double-Stage Convergent Methods, Z. Xu, M. Kahr, K.
`L. Walker, C. L. Wilkins, and 1. S. Moore, J. Am. Chern.
`Soc., 1994, 116, 4537-50.
`
`Conjugated Macromolecules of Precise Length and Consti(cid:173)
`tution. Organic Synthesis for the Construction of Nanoar(cid:173)
`chitectures, 1. M. Tour, Chern. Rev., 1996, 96, 537-53.
`
`A Molecular Photonic Wire, R. W. Wagner, and 1. S.
`Lindsey, 1. Am. Chern Soc., 1994, 116, 9759-60.
`
`Boron-dipyrromethene Dyes for Incorporation in Synthetic
`Multi-pigment Light-harvesting Arrays, R. W. Wagner, and
`1. S. Lindsey, Pure & Appl. Chern., 1996, 68, 1373-80.
`
`Heterodimeric DNA-binding dyes designed for energy
`transfer: synthesis and spectroscopic properties, S.C. Ben(cid:173)
`son, P. Singh, AN. Glazer, Nucleic Acids Res. 1993, 2I,
`5727-35.
`
`New energy transfer dyes for DNA sequencing, L.G. Lee,
`S.L. Spurgeon, C.R. Heiner, S.C. Benson, B.B. Rosenblum,
`S.L. Menchen, R.1. Graham, A. Constantinescu, K.G.
`Upadhya, 1.M. Cassel, Nucleic Acids Research 1997, 25,
`2816-22.
`
`Difiuorboryl-Komplexe von Di- und Tripyrrylmethenen, A
`Treibs, F.H. Kreuzer, Liebigs Ann. Chern. 1968, 718,
`208-23. BODIPY = 4,4-difiuoro-4-bora-3a,4a-diaza-s(cid:173)
`indacene.
`
`Synthesis of 2, 6-Diethyl-3-methacroyloxymethyl-1, 5, 7,
`8-tetramethylpyrromethene-BF2 for the Preparation of New
`Solid-State Laser Dyes, T. Chen, 1.H. Boyer, M.L. Trudell,
`Heteroatom. Chern. 1997, 8, 51-4.
`
`Directed Electrophilic Cyclizations: Effecient Methodology
`for the Synthesis of Fused Polycyclic Aromatics, M.B.
`Goldfinger, K.B. Crawford, T.M. Swager, J. Am. Chern. Soc.
`1997, 119, 4578-4593.
`
`and
`Anthryloliogothienylporphyrins: Energy Transfer
`Light- Harvesting Systems, M.S. Vollmer, F. Wurthuer, F.
`Effenberger, P. Emele, D.U. Meyer, e. al, Chern. Eur.. J.
`1998, 4, 260--9.
`
`Steroid-Bridged Anthryloligothienylporphyrins: Synthesis
`and Study on the Intramolecular Energy Transfer, M.S.
`Vollmer, F. Effenberger, T. Stumpfig, A Hartschuh, H. Port,
`H.C. Wolf, J. Org. Chern. 1998, 63, 5080-7.
`
`* cited by examiner
`
`2
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 1 of 13
`
`US 6,340,750 Bl
`
`a Through space FET from a donor dye
`Figure 1.
`D to an acceptor dye A;
`b through bond FET.
`
`a through space FET
`
`b through bond FET
`
`hv' /
`
`FIG. 1A
`
`FIG. 18
`
`3
`
`

`

`U.S. Patent
`
`Jan. 22, 2002
`
`Sheet 2 of 13
`
`US 6,340,750 Bl
`
`0 0
`
`0
`
`1
`R2
`aa Rl - R2
`== a
`ab Rl== a, R2
`== b
`
`FIG. 2A
`
`2
`R2
`aa Rl - R2 = a
`ab Rl== a, R2
`== b
`
`FIG. 28
`
`4
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 3 of 13
`
`US 6,340,750 Bl
`
`II
`
`FIG. 2C
`
`II
`
`Et
`
`Et
`
`FIG. 2D
`
`5
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 4 of 13
`
`US 6,340,750 Bl
`
`()____
`
`N
`H
`
`FIG. 3A
`
`a, b
`--•.., R
`
`R
`
`a R = H
`b R = Et
`
`H
`
`II
`
`c, d
`-----~~~ R
`
`R
`
`FIG. 38
`
`6
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 5 of 13
`
`US 6,340,750 Bl
`
`(yoo
`}r-N"',
`0
`0
`
`I
`
`e, f
`----~laa and lab
`
`5
`
`Br
`
`FIG. 3C
`
`(yoo
`}r-N"'
`0
`0
`
`I
`
`g, h
`_ _ .,.... 2aa and 2ab
`
`Br
`6
`Scheme 1. Syntheses of the cassettes 1 and 2, a)
`CH2Cl2 reflux: b) BF3•0Et2, NEt3, MePh, 80 °C, 26%
`steps) for 3a and 39%
`(2 steps) for 3b; c) HCCTMS,
`NEt3, cat. Pd(PPh3)4, cat. Cui, MePh 60 °C, 99% for a
`and 96% for b; d) TBAF, THF, 0 °C, 60% for a and 58%
`forb; e) ( 4a, NEt3, cat. Pd(PPh3)4, cat. Cui, MePh
`50 °C, 96%; f)4a or 4b, NEt3, cat. Pd(PPh3)4, cat.
`Cui, MePh 80 °C, 65% for laa and 23% for lab; g) 4a,
`NEt3, cat. Pd(PPh3)4, cat. Cui, MePh 45 °C, 83%; f)
`4a or 4b, NEt3, cat. Pd(PPh3)4, cat. Cui, MePh 80°,
`65% for laa and 17% for lab.
`
`(2
`
`FIG. 3D
`
`7
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 6 of 13
`
`US 6,340,750 Bl
`
`Important spectroscopic data for
`Table 1.
`compounds 4, and the cassettes 1 and 2.
`
`(abs) a
`Xffiax
`(nm)
`
`(ems) b
`Xffiax
`(nm)
`
`4a
`4b
`laa
`lab
`2aa
`2ab
`
`504
`529
`504
`and 529
`504
`and 529
`
`505
`
`505
`
`515
`543
`515
`542
`516
`543
`
`energy
`transfer (ET)
`efficiency b,c
`9-
`0
`
`>90
`
`>90
`
`ratios of
`fluorescence
`intensitiesc
`
`4a
`4b
`laa
`lab
`2aa
`2ab
`
`laa:4a 1.5: 1.0
`lab:4b 2.2: 1.0
`2aa:4a 1.6: 1.0
`2ab:4b 1.7: 1.0
`[b] where ET = {1 -
`[a] in CHCl3.
`(fluorescence
`intensity of donor emission in cassette)/(fluorescence
`intensity of donor alone)} x 100% [c] excitation at 488
`nm.
`
`F/6.4
`
`8
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 7 of 13
`
`US 6,340,750 Bl
`
`Br
`
`acceptor
`
`donor
`
`la-e
`
`FIG5A
`
`acceptors:
`
`Rl
`a Me
`b Me
`c H
`
`Et
`H
`
`Me
`2-Me0C6H4
`
`FIG. 58
`
`9
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 8 of 13
`
`US 6,340,750 Bl
`
`acceptors:
`
`FIG. 5C
`
`R4
`H
`OMe
`
`d
`e
`
`4a
`
`FIG. 6
`
`10
`
`

`

`U.S. Patent
`
`Jan. 22,2002
`
`Sheet 9 of 13
`
`US 6,340,750 Bl
`
`120~~~~~~~~~~~~~
`
`100 1 - - - -+ - -w - - -+ - - -+ - - - - -+ - - -+ - - - - - 1
`~ 80~--~~~--~--~--~--~
`s:::
`~ 60r---~~~--~---+---+--~
`s
`~ 40r---++--~+-hH---+---+--~
`~
`
`20~----+~~~~~~-~~
`
`o~--~~~~~~~~~ .. ~
`450
`500
`550
`600
`650
`700
`750
`FIG. lA
`
`120~~~~~~~~~~~~~
`
`100 1----+-----+---..----+-.....----+-.....---+----t
`~ 80~--~~~~~~~~~--~
`fg
`~ 60~--~~~~++~~~~--~
`.s:
`' 40~--~~~~~~~--~--~
`~
`
`20~--~--~--~--~--~--~
`
`o~~~~~~~~~~~~~
`450
`500
`550
`600
`650
`700
`750
`Wavelength (nm)
`FIG. 78
`
`11
`
`

`

`U.S. Patent
`
`Jan. 22,2002
`
`Sheet 10 of 13
`
`US 6,340,750 Bl
`
`100~~~~~~~~~~~~~
`
`eo~~~-----+----~--~~~
`
`~60~~~~--~~--~~~~~
`~40H-~~~--~~~~~--~~
`.s
`20 11-----f-~r--~r-:-1--~ h---h'----lr---+-1\--1
`
`0
`500
`
`650
`600
`550
`Wavelength (nm)
`FIG. BA
`
`700
`
`oo~--~----~--~~~~
`
`~60~~~~--~~--~~~~~
`(I) a;
`.s
`20 .___---J--~-~......,._._ __ __;;,.!1 lh---h'----4----+-A---1
`
`~4oH-~~~--~-4-4~~--~~
`
`0
`500
`
`650
`600
`550
`Wavelength (nm)
`FIG. 88
`
`700
`
`12
`
`

`

`U.S. Patent
`
`Jan. 22, 2002
`
`Sheet 11 of 13
`
`US 6,340,750 Bl
`
`100
`
`1- I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`J
`
`I
`
`'
`
`I
`
`I
`
`I I ' '-
`_I
`676 nm :
`515nm
`I' .._160 nm_,. )
`--
`\ \
`~-.~ I
`
`~
`
`1a
`1e
`
`300
`
`1-
`1-
`
`80
`
`-----------.
`
`,
`
`~60
`
`~ ...... .s 40
`20
`
`0
`
`\ -
`~ I
`.._}I I
`600
`500
`400
`700
`Wavelength (nm)
`FIG. BC
`
`------
`
`I
`
`I I' '-
`.I
`676 nm :
`515nm
`r r---160 nm (\
`-
`...,..,
`-
`-
`-
`-
`-
`--
`-
`\ '
`\ -
`'~
`700
`
`\
`
`J
`I
`I
`
`I
`
`I
`
`'
`I
`I
`\
`
`' I
`I '
`
`I
`
`\
`
`I
`
`I
`
`I
`
`' t
`I :
`\
`:
`' l
`l
`I I
`~ ~-.~ I
`.JM .. l I
`400
`500
`600
`Wavelength (nm)
`FIG. BD
`
`1a
`-----1 e
`
`300
`
`1--
`~60
`---
`fll
`.!! .s 40
`---
`20
`
`--.
`
`....
`
`0
`
`100
`
`;.... I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`1-
`1-
`
`1-
`
`80
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`13
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 12 of 13
`
`US 6,340,750 Bl
`
`~ j
`
`Br
`
`(i)HCCSiMe3, cat. Pd(PPH3)4/CuL
`THF, NEt3, 12 h, 55° C
`54%
`
`Br
`Br
`(ii) TBAF, THF, -78° C, 10 min
`93%
`
`~ j
`
`3
`
`FIG. 9A
`
`2a-e, cat. Pd(PPh3) 4 /CuL
`
`3
`
`NEt3, THF, 55° C, 12 h
`
`la-e
`83-97%
`
`FIG. 98
`
`I
`
`FIG. 9C
`
`2a-c
`
`14
`
`

`

`U.S. Patent
`
`Jan.22,2002
`
`Sheet 13 of 13
`
`US 6,340,750 Bl
`
`I
`
`FIG. 90
`
`2d-e
`
`DNA
`linking
`group
`
`donor
`
`1
`
`FIG. 10
`
`acceptor
`
`15
`
`

`

`US 6,340,750 Bl
`
`1
`THROUGH BOND ENERGY TRANSFER IN
`FLUORESCENT DYES FOR LABELLING
`BIOLOGICAL MOLECULES
`
`2
`rescence is more efficient than other systems wherein two
`identical fluorescent labels per primer have been used to
`enhance sensitivity.[ A14]
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims the benefit of U.S. Provisional
`Patent Application entitled "Through Bond Energy Transfer
`in Fluorescent Dyes for Labelling Biological Molecules,"
`Ser. No. 60/112,711 filed Dec. 18, 1998.
`
`RIGHTS IN THE INVENTION
`
`5
`
`The utility of the dye-terminator approach has also been
`enhanced, but in this case the development was one in
`molecular biology. Tabor and Richardson showed that some
`mutated DNA polymerases favor incorporation of labeled
`ddNTPs. [ A15] These enzymes are more expensive than the
`10 wild type, but they can be obtained in significant quantities
`via over-expression. Use of these DNA replicating enzymes
`leads to more efficient use of ddNTPs in Sanger sequencing,
`and this is particularly important when the ddNTP bears a
`label.
`
`This invention was made in part with United States
`Government support under grant number HG01745 awarded 15
`by the National Institute of Health, and the United States
`Government has certain rights in the invention.
`
`The state of the art in high throughput sequencing is such
`that both dye-primers and dye terminators are used.
`Typically, cloned genomic fragments are randomly sheared
`and subcloned into specialized sequencing vectors, i.e., the
`20 "shot-gun" approach. Doubly labeled dye-primers that
`complement the specialized vector arms are then used to
`begin the sequencing operation; a compelling advantage of
`this is that only a limited repertoire of these expensive
`primers is required. Primer walking is then used to extend
`25 the sequence information obtained. However, the primer
`walking steps, and sequencing of regions riot-covered by the
`shot-gun/primer walking process, require primers that are
`tailor made to those particular sequences (rather than to a
`restriction site sequences). Syntheses of many different
`30 doubly labeled dye-based primers cannot be justified, so a
`different approach is used. In fact, it is cost effective to use
`labeled ddNTPs/mutant DNA replicating enzymes at this
`stage, therefore obviating the need for extensive dye-primer
`syntheses.
`
`TECHNICAL FIELD OF THE INVENTION
`
`The present invention relates in general to the field of
`DNA sequencing and, more particularly, to through bond
`energy transfer in fluorescent dyes for labelling biological
`molecules.
`
`BACKGROUND OF THE INVENTION
`
`Methods routinely applied for high throughput DNA
`sequencing have oscillated between two embodiments of the
`Sanger scheme. [ A1, A2] Fluorescence detection dominates
`throughout, [ A3] but the factor that distinguishes the
`approaches is that the labels can be situated in the primer
`(dye-primers) or in the terminating fragments (dye(cid:173)
`terminators). Both methods have been, and continue to be,
`used. [A4-A6]
`Early dye-primer technology featured one fluorescent flag 35
`per primer. Four reactions were performed with each of the
`ddNTP's using the "workhorse tags", i.e., JOE, TAMRA,
`ROX, and FAM. These four reactions were mixed after
`production of a nested set of chain terminated DNA
`fragments, and analyses were performed via gel electro- 40
`phoresis in one lane with a static detector.
`Dye terminator strategies [A7] have the advantage that
`only one reaction is required to produce a nested set of chain
`terminated DNA fragments labeled with fluorescent groups
`appropriate to the four ddNTP's. The (unlabeled) primers 45
`used are also cheaper to produce than the corresponding
`fiuorescently labeled ones. Moreover, in contrast to dye(cid:173)
`primer strategies, pausing bands are invisible to fluorescence
`detection when the label is present only in the terminator.
`The disadvantage of dye-terminators is that not all of the 50
`relatively precious labeled component is incorporated into
`the complement whereas all the fluorescence is retained in
`the complement if the dye primer method is used.
`A significant advance in dye-primer methodology
`occurred when it was realized that the fluorescence signal
`could be enhanced by approximately ten-fold when two
`labels were used in the following way. [A9] One was
`selected to absorb relatively high energy photons; energy
`transfer though space to the second fluorescent group would
`then lead to emission at a lower wavelength. Specifically,
`FAM was (and is) used to harvest the irradiation, then
`convey energy through space to either JOE, TAMRA, or
`ROX. The ten-fold enhancement obtained is significant
`because it facilitates use of less reagents (dye-primer,
`enzyme, dNTPs, ddNTPs, etc.), and/or lessens the need to
`concentrate the reactions before gel electrophoresis and
`detection.[A9-A13] Energy transfer enhancement of fino-
`
`SUMMARY OF THE INVENTION
`
`Fluorescent energy transfer cassettes are reported. Unique
`features of these are that they allow through bond energy
`transfer and have a succinimidyl ester functionality suitable
`for attaching them to biomolecules. The relevance of this
`design concept to high throughout DNA sequencing is
`discussed.
`
`This disclosure outlines a general design principle for new
`fluorescent dyes to be applied in high throughput DNA
`sequencing protocols (e.g., The Genome Project) and other
`applications in biotechnology.
`
`Fluorescent dyes for DNA sequencing and other biotech(cid:173)
`nological applications can be produced in the following way.
`A UV-absorbing chromophore is selected that will absorb
`relatively strongly at the wavelength emitted by the source
`chosen for the application under consideration. Organic
`synthesis is then performed to incorporate this chromophore
`55 into a molecule wherein the chromophore is conjugated with
`a molecular entity having desirable fluorescence emission
`properties. In DNA sequencing, the latter group would be
`one with a strong, narrow bandwidth, emission at a distinctly
`different wavelength to the other dyes used in the sequencing
`60 method. The UV chromophore must absorb at a lower
`wavelength than the fluorescence emitter, and it is highly
`desirable that the chromophore and fluorescence emitter be
`placed at opposite ends of the conjugated system (not in the
`middle). In the anticipated mode of action of these dyes, the
`65 UV absorbing group would harvest radiation from the exci(cid:173)
`tation source and transmit it through the conjugated system
`to the fluorescence emitter which would then fluoresce.
`
`16
`
`

`

`US 6,340,750 Bl
`
`4
`FIG. 3 is a diagram illustrating synthesis of cassettes 1
`and 2. a) CH2 CL2 reflux: b) BF3 .0Et2 , NEt3 , MePh, 80° C.,
`26% (2 steps) for 3a and 39% (2 steps) for 3b; c) HCCTMS,
`NEt3 , cat. Pd(PPh3 ) 4 , cat. Cul, MePh 60° C., 99% for a and
`5 96% forb; d) TABF, THF, 0° C., 60% for a and 58% forb;
`e) 4a, NEt3 , cat Ph(PPh3 ) 4 , cat. Cul, MePh 50° C., 96%; f)
`4a or 4b, NEt3 , cat. Pd(PPh3 ) 4 , cat. Cul, MePh 80° C., 65%
`for laa and 23% for lab; g) 4a, NEt3 , cat. Pd(PPh3 ) 4 , cat.
`Cul, MePh 45° C., 83%; f) 4a or 4b, NEt3 , cat. Pd(PPh3 ) 4 ,
`10 cat. Cul, MePh 80° C., 65% for laa and 17% for lab; and
`FIG. 4 is a table illustrating important spectroscopic data
`for compounds 4, and the cassettes 1 and 2.
`FIGS. SA, SB, and SC depict donor and acceptor portions
`of the fluorescent dyes. FIG. SA shows the anthracene
`derivative donors, while FIGS. SB and SC show the
`BODIPY acceptor units.
`FIG. 6 depicts a dye suitable for coupling to DNA
`FIG. 7A shows that current dyes for DNA sequencing
`have overlapping fluorescence emissions spanning approxi(cid:173)
`mately 80 nm and decreasing intensities at longer wave(cid:173)
`lengths.
`FIG. 7B shows that ideal dyes would be better resolved
`and fluoresce strongly with equal intensities.
`FIG. SA shows the normalized fluorescence emission
`spectra (270 nm excitation) of 1 ,uM chloroform solutions of
`dyes la-e.
`FIG. 8B shows the normalized fluorescence emission
`spectra (270 nm excitation) of 1 ,uM chloroform solutions of
`dyes la and le.
`FIGS. 9A-D depicts the synthesis of dyes la-e.
`FIG. 9Ashows the conversion of 9,10-dibromoanthracene
`to compound 3.
`FIG. 9B shows the linking of compound 3 to compounds
`2a-e to afford dyes 1 a-e.
`FIG. 9C shows the chemical structures of compounds
`2a-c, and
`FIG. 9D shows the chemical structures of compounds
`40 2d-e.
`FIG. 10 shows a dye molecule containing a DNA linking
`group. The dye can capture light from a blue laser and emit
`at much longer wavelengths.
`
`20
`
`35
`
`15
`
`3
`The new fluorescent dyes should also preferably have the
`following properties:
`(i) manageable solubility characteristics;
`(ii) functionality that allows them to be conveniently
`attached to nucleotides (or other biomolecules);
`(iii) similar structures when used as sets for DNA
`sequencing, thus giving near tagged DNA fragments
`with similar gel mobilities;
`(iv) chemical stability;
`(v) chemical accessibility (i.e., can be obtained via con(cid:173)
`venient syntheses); and,
`(vi) functional groups which facilitate convenient and
`economical incorporation of the labels.
`According to one embodiment, the design principle dis-
`closed here provides dyes that can be designed to:
`harvest radiation (from lasers and similar devices) in
`regions of the electromagnetic spectrum that cannot be
`efficiently absorbed by the dyes currently used for DNA
`sequencing, thus allowing a wider variety of light
`source wavelengths to be used;
`fluoresce in a greater wavelength range than the four dye
`detection system most often used at present (i.e., JOE,
`TAMRA, ROX, FAM) allowing greater resolution of
`the fluorescence emission from the dyes giving a more 25
`accurate read in DNA sequencing experiments;
`give more intense fluorescent emission on irradiation with
`a usable source than is currently possible using JOE,
`TAMRA, ROX, and FAM, thus giving increased sen(cid:173)
`sitivity and enabling smaller amounts of samples to be 30
`detected;
`give fluorescence emission from a usable source that is
`comparable or superior to the through space energy
`transfer dyes introduced by Mathies, and by Gibbs, and
`their coworkers;
`be introduced more conveniently and economically than
`the through space energy transfer dyes introduced by
`Mathies, and by Gibbs, and their co-workers; and,
`be useful in both the "dye-primer" and the "dye(cid:173)
`terminator" approaches to DNA sequencing.
`Sets of fluorescent dyes would be prepared such that one
`UV absorbing group was paired with four different fluores(cid:173)
`cent emitter moieties, each with clearly different emission
`wavelengths. This would allow strong fluorescence at four
`clearly distinguishable wavelengths.
`There is also potential for two different sets of sequencing
`reactions to be mixed and analyzed in a single gel electro(cid:173)
`phoresis run. Thus, if two UV absorbing molecules that
`absorbed in mutually exclusive regions of the spectrum were
`each paired with four dyes, emission would only occur in 50
`one set if the absorbance were tuned to one UV absorbing
`group. Alternatively, eight different dyes could be coupled
`with one or two UV absorbing groups (four each) to achieve
`the same end.
`
`45
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The preferred embodiments of the present invention and
`its advantages are best understood by referring to the FIGS.
`1 through 4 of the drawings according to the teachings of the
`invention.
`Several conclusions may be made based on the discus(cid:173)
`sions above. First, enhancement of fluorescence emission is
`desirable. Second, both dye-primer and dye-terminator
`approaches are viable, and the selection of one over the other
`55 is not a clear cut choice. It is possible, for instance, that if
`the dye-terminator methodology were improved then most
`sequencing reactions might be done that way. If they were,
`the number of reactions necessary to generate DNA comple(cid:173)
`ments would be reduced by a factor of four relative to
`60 dye-primer approaches (since the four different ddNTP's can
`be mixed), and pausing bands would become invisible.
`There are at least two ways to improve the utility of the
`dye-terminator approach. Energy transfer emission
`enhanced fluorescent tags for ddNTP's have not yet been
`65 developed, so work in this area is very likely to be useful.
`Another avenue to explore is to devise completely new
`fluorescent labels for ddNTP's.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`A more complete understanding of the invention and its
`advantages will be apparent from the detailed description
`taken in conjunction with the accompanying drawings in
`which:
`FIGS. 1a and 1b are schematic diagrams illustrating
`energy transfer "through space" and energy transfer
`"through bonds," respectively, for the production of fluo(cid:173)
`rescent labels for biological systems;
`FIG. 2 is a diagram illustrating the structures of four
`cassettes used according to the teachings of the present
`invention for labeling DNA or other biological molecules;
`
`17
`
`

`

`US 6,340,750 Bl
`
`6
`ponents. This coupling reaction is more efficient than the
`Wadsworth-Emmons reaction generally used to produce the
`corresponding systems with alkene rather than aryne link(cid:173)
`ages. In fact, solid phase syntheses of these materials are
`5 possible as a direct consequence of the efficiency of the
`Sonogashira coupling.
`
`5
`Superior dye-primer labels must overcome the false prim(cid:173)
`ing and mobility shift problems, and the experimental incon(cid:173)
`veniences associated with label incorporation. When singly
`labeled dye primers are used, mobility differences are com(cid:173)
`pensated by virtual corrections to the data after detection but
`prior to output of the read. Almost all of the work with
`double dye primers involves labels supported on T-analogs
`which constitute part of the primer sequence. Those systems
`can be vulnerable to false priming due to these unnatural
`nucleotides. Moreover, the mobility correction varies with 10
`sequence and cannot always be adequately accommodated
`by virtual corrections. To address this problem, Mathies and
`co-workers designed a system that they term "cassette
`labeling". [A16] In this method, the primer syntheses are 15
`performed in such a way that the first fluorescent base is
`added at the 5'-termini of the primers, then six more cycles
`of phosphoramidite couplings are performed using deoxyri(cid:173)
`bose units with no purine or pyrimidine functionalities.
`Finally, the other label is added at the end of this chain. The 20
`primers formed in this way are less vulnerable to false
`priming, and the DNA complements derived from them have
`improved mobility characteristics and exhibit less fluores(cid:173)
`cence quenching. However, this strategy is not ideal for
`several reasons. Most important of these are the fact that 25
`these primers require seven more coupling steps than are
`required to generate the primer sequence. The word "cas(cid:173)
`sette" is inappropriate for this system because it implies that
`the dye labels with the appropriate spacing are simply
`slotted in; in fact, they are built on the end of the primer in 30
`a multi-step operation which must be repeated for each
`primer. Second, the linker between the two labels is flexible.
`Consequently, the fluorescence emission will be the result of
`averaged conformational states which may vary according to 35
`the different environments of the label system. Third, the
`radiation that can be used to excite the labels must be chosen
`within relatively restrictive wavelength regions (e.g. 488 nrn
`or 154 nrn source, but not ones much lower in wavelength)
`. An eight dye system with four responding to one excitation 40
`wavelength and four responding at another would be
`extremely hard or almost impossible to develop given the
`dyes available. Finally, the issue of gel mobilities is not a
`solved problem because different conformational states may
`still be present in ratios that vary with the peripheral primer 45
`sequence. Energy transfer systems based on BODIPY dyes
`have been introduced for enhanced sensitivity and improved
`gel mobility factors in DNA sequencing, [ A17] but the
`concerns outlined above still apply. [ A18]
`
`OR
`
`Ph
`
`II
`
`Ph
`
`Extended aryl alkyne molecules are not particularly
`soluble in any common solvent, but the alkoxide subsituents
`shown in structure I can be used to give appreciable solu(cid:173)
`bilities. In nearly all literature on these compounds the "OR"
`functionalities are 0-hydrocarbons included for compatibil(cid:173)
`ity with organic media, although in at least one case a water
`soluble system has been produced when R was a sulfonated
`benzylic group.
`Molecules of type I are chemically robust. They would
`not, for instance, react or decompose under the thermal
`cycling conditions used for enzymatic generation of DNA
`50 components.
`The spacing between repeat units in the polymers I is
`approximately 6.75 A. This rigidity could be exploited to
`hold a UV absorbing group and a fluorescence emitter at a
`relatively invariant and easily estimated separation.
`Photophysical properties of framework I are such that
`absorption occurs at around 448 nm and fluorescence emis(cid:173)
`sion occurs at 474 nm (n=-22). High fluorescent quantum
`yields are often observed (ca 0.8 to 0.9 for many molecules
`of type I) presumably because the rigidity of the system
`60 precludes bond motions that would otherwise result in
`radiationless decay. Moreover, the emission spectrum tends
`to be relatively sharp, much sharper than the absorption
`spectrum. The most relevant property of these materials to
`this project is the energy transfer properties seen for mol-
`65 ecules like polymer II. This material emits at 524 nm
`irrespective of the wavelength of the absorption. It appears
`that random excitation of the polymer backbond transmits
`
`55
`
`Dye-primer methodologies may be improved by generat(cid:173)
`ing a double-dye cassette that could be conveniently incor(cid:173)
`porated into a primer in one step. This cassette is preferably
`relatively rigid to minimize sequence dependent mobility
`variations.
`
`No compounds of the this type (i.e., fluorescent com(cid:173)
`pounds having a UV-harvesting group in c