Published on Web 02/03/2006
`
`Design and Synthesis of a Chemically Cleavable Fluorescent Nucleotide,
`3¢-O-Allyl-dGTP-allyl-Bodipy-FL-510, as a Reversible Terminator for DNA
`Sequencing by Synthesis
`Lanrong Bi,†,‡ Dae Hyun Kim,†,§ and Jingyue Ju*,†,‡
`Columbia Genome Center, Columbia UniVersity College of Physicians and Surgeons,
`New York, New York 10032, and Departments of Chemical Engineering and
`Biomedical Engineering, Columbia UniVersity, New York, New York 10027
`
`Received October 20, 2005; E-mail: ju@genomecenter.columbia.edu
`
`With the completion of the human genome project, there is now
`a focus on developing new DNA sequencing technology that will
`dramatically reduce the cost of sequencing without sacrificing
`accuracy. This will ultimately enable personalized medicine in
`healthcare.1 Current state-of-the-art DNA sequencing technologies
`face limitation in terms of cost, read length, and throughput. DNA
`sequencing by synthesis (SBS), where the identity of each nucleo-
`tide is detected immediately after its incorporation into a growing
`strand of DNA in a polymerase reaction, offers an alternative
`approach to address some of these limitations. An important
`requirement for the SBS approach is a 3¢-OH-capped fluorescent
`nucleotide that can act as a reversible terminator.2 Following the
`identification of the nucleotide incorporated in a DNA polymerase
`reaction, the 3¢-OH capping group along with a fluorescent label
`are removed to regenerate a free 3¢-OH group, thus allowing DNA
`chain elongation. The importance of removing the fluorescent label
`after each base identification is to ensure that
`the residual
`fluorescence from the previous nucleotide incorporation does not
`affect
`the identification of the next
`incorporated fluorescent
`nucleotide. We have reported the design and synthesis of a 3¢-O-
`allyl photocleavable fluorescent nucleotide analogue, 3¢ -O-allyl-
`dGTP-PC-Bodipy-FL-510, as a reversible terminator to determine
`two repeated nucleotide sequences in SBS.3 A two-step process,
`photocleavage and Pd-catalyzed deallylation, was used to remove
`the fluorophore and the 3¢-O-allyl group, respectively. 3,4
`The speed and sequence read length of SBS depend on the
`efficiency of the fluorophore and allyl group cleavage reactions.
`Due to multiple steps required in the identification, removal of the
`fluorescent label, and regeneration of the 3¢-OH group after each
`nucleotide incorporation in SBS, even minor losses in efficiency
`at each step may lead to inhibited read length. For this reason, any
`improvement in efficiency within each cycle of nucleotide identi-
`fication, fluorophore removal, and 3¢-OH regeneration can have
`significant impact on read length, thus tackling the physical limit
`in DNA sequencing by synthesis.
`A disulfide linker has been previously explored as a chemically
`cleavable moiety to attach a fluorophore to a deoxynucleotide, and
`the use of 2-mercaptoethanol to remove the fluorophore after the
`nucleotide incorporation and detection in SBS has also been
`studied.5 However, the disulfide bond can be reversed and destab-
`ilized under certain conditions.6,7 Encouraged by our successful
`application of allyl protection of the 3¢ -OH group of the nucleotide
`analogues,3,4 we have explored the construction of a novel chemi-
`cally cleavable fluorescent labeling system based on an allyl group
`to modify a nucleotide. The goal was to discover linker chemistry
`
`† Columbia University College of Physicians and Surgeons.
`‡ Department of Chemical Engineering, Columbia University.
`§ Department of Biomedical Engineering, Columbia University.
`2542 9 J. AM. CHEM. SOC. 2006, 128, 2542-2543
`
`Scheme1. Synthesis of 3¢-O-Allyl-dGTP-allyl-Bodipy-FL-510 ( 4)
`
`and condition to remove the fluorophore and the 3¢-O-allyl group
`in one step to increase the SBS efficiency. We report here that an
`allyl moiety can be used successfully as a linker to tether a fluoro-
`phore to a 3¢-O-allyl-modified nucleotide, forming a chemically
`cleavable reversible terminator, 3¢ -O-allyl-dGTP-allyl-Bodipy-FL-
`510 4 (Scheme 1).8 We have found that the fluorophore and the
`3¢-O-allyl group on a DNA extension product, which is generated
`by incorporation of 4, are removed simultaneously in 30 s by Pd-
`catalyzed deallylation in aqueous solution. This one-pot dual-deallyla-
`tion reaction thus allows the reinitiation of the polymerase reaction.
`Here, we describe the design and synthesis of a nucleotide analogue,
`3¢ -O-allyl-dGTP-allyl-Bodipy-FL-510 4, with a fluorophore attached
`to the 7 position of a guanine base via an allyl carbamate linker,
`and its application as a reversible terminator for SBS.
`Readily available allylic alcohol 1 was chosen as a starting
`material for the preparation of 4. First, allylic alcohol 1 was reacted
`with N-hydroxysuccinimide (NHS) ester of the BODIPY-FL-510
`to produce allylic-Bodipy-FL-510-NHS 2, which was subsequently
`converted to its corresponding NHS ester 3 by reacting with N,N¢-
`disuccinimidyl carbonate. The coupling reaction between 3 and the
`modified nucleotide (3¢-O-allyl-dGTP-NH 2)3 produced the chemi-
`cally cleavable fluorescent nucleotide, 3¢-O-allyl-dGTP-allyl-Bo-
`dipy-FL-510 4.
`
`10.1021/ja057136n CCC: $33.50 © 2006 American Chemical Society
`
`Illumina Ex. 1085
`IPR Petition - USP 10,435,742
`
`

`

`Scheme2. Polymerase DNA Extension Reaction Using
`3¢ -O-Allyl-dGTP-allyl-Bodipy-FL-510 as a Reversible Terminator
`
`C O M M U N I C A T I O N S
`
`Figure 1. A polymerase extension scheme (left) and MALDI-TOF MS
`spectra of extension and dual-deallylation product (right).
`
`corresponding to a DNA product 6 with both the fluorophore and
`3¢ -O-allyl removed, appeared as the sole product. The absence of
`a peak at m/z 6967 confirms that the one-pot dual-deallylation
`reaction to remove both the fluorophore and the 3¢ -O-allyl group
`from the DNA product was very efficient. Figure 1C shows that
`DNA product 6 can be successfully used as a primer to continue
`the incorporation of another nucleotide 4 to yield product 7 (m/z
`7348). Upon deallylation, both the fluorophore and the 3¢ -O-allyl
`group of 7 were removed quantitatively to yield 8 (m/z 6893).
`Thus, these experimental results demonstrate that the allyl moiety
`on the nucleotide analogue is completely stable in a polymerase
`extension condition and can be selectively cleaved in a rapid and
`efficient manner. Furthermore, the nucleotide analogue, 3¢ -O-allyl-
`dGTP-allyl-Bodipy-FL-510, can be faithfully incorporated into a
`growing DNA strand in a polymerase extension reaction to act as
`a reversible terminator in SBS. As a result, we expect expansion
`of this novel linker and protection strategy to other applications
`that include bioconjugation and solution- and solid-phase organic
`synthesis.
`Acknowledgment. We thank New England Biolabs for provid-
`ing the 9(cid:176) N DNA polymerase. This work was supported by NIH
`Grants P50 HG002806 and R01 HG003582 and the Packard
`Fellowship for Science and Engineering.
`Supporting Information Available: Experimental procedures,
`characterization data. This material is available free of charge via the
`Internet at http://pubs.acs.org.
`
`References
`
`(1) Collins, F. S.; Green, E. D.; Guttmacher, A. E.; Guyer, M. S. Nature
`2003, 422, 835-847.
`(2) Ju, J.; Li, Z.; Edwards, J.; Itagaki, Y. U.S. Patent 6,664,079, 2003.
`(3) Meng, Q.; Kim, D. H.; Bai, X.; Bi, L.; Turro, N. J.; Ju, J. J. Am. Chem.
`Soc. Submitted
`(4) Ruparel, H.; Li, Z.; Bai, X.; Kim, D. H.; Turro, N. J.; Ju, J. Proc. Natl.
`Acad. Sci. U.S.A. 2005, 102, 5932-5937.
`(5) Mitra, R. D.; Shendure, J.; Olejnik, J.; Olejnik, E. K.; Church, G. M.
`Anal. Biochem. 2003, 320, 55-65.
`(6) Pleasants, J. C.; Guo, W.; Rabenstein, D. L. J. Am. Chem. Soc. 1989,
`111, 6553-6558.
`(7) Huyghues-Despointes, B. M. P.; Nelson, J. W. Biochemistry 1992, 31,
`1476-1483.
`(8) See the Supporting Information for experimental procedures and charac-
`terization data for all the compounds in Schemes 1 and 2.
`
`JA057136N
`
`J. AM. CHEM. SOC. 9 VOL. 128, NO. 8, 2006 2543
`
`To verify that 3¢ -O-allyl-dGTP-allyl-Bodipy-FL-510 4, acting
`as a reversible terminator, is incorporated accurately in a base-
`specific manner in a polymerase reaction, we have performed a
`polymerase DNA extension reaction, as shown in Scheme 2.8 This
`allows the isolation of the DNA product at each step for detailed
`molecular structure characterization by using MALDI-TOF MS,
`as shown in Figure 1. First, a polymerase extension reaction using
`4 as a terminator along with a primer and the synthetic 100-mer
`DNA template corresponding to a portion of exon 7 of the human
`p53 gene was performed to yield a single-base extension product
`5. After the reaction, a small portion of the extension product 5
`was characterized by MALDI-TOF MS. The rest of the extended
`DNA product 5 was added to a deallylation cocktail [1X Thermopol
`reaction buffer/Na2PdCl4/P(PhSO3Na)3] and incubated for 30 s to
`yield deallylated DNA product 6, which was characterized by
`MALDI-TOF MS.
`The deallylated DNA product with both the fluorophore removed
`and a free 3¢ -OH group regenerated can then be used as a primer
`for the next nucleotide extension reaction.
`Figure 1 (right panel) shows the sequential mass spectrum at
`each step of DNA sequencing by synthesis using 3¢ -O-allyl-dGTP-
`allyl-Bodipy-FL-510 4 as a reversible terminator. As can be seen
`from Figure 1A, the MALDI-TOF MS spectrum consists of a
`distinct peak at m/z 6967, corresponding to the single-base DNA
`extension product 5 with 100% incorporation efficiency, confirming
`that the reversible terminator 4 can be incorporated base-specifically
`by DNA polymerase into a growing DNA strand. Figure 1B shows
`the one-pot dual-deallylation result after 30 s incubation of the DNA
`extension product in a deallylation cocktail solution. The peak at
`m/z 6967 completely disappeared, whereas the peak at m/z 6512,
`
`