Design and synthesis of a 3ⴕ-O-allyl photocleavable
`fluorescent nucleotide as a reversible terminator
`for DNA sequencing by synthesis
`
`Hameer Ruparel*†, Lanrong Bi*†, Zengmin Li*†, Xiaopeng Bai*†‡, Dae Hyun Kim*§, Nicholas J. Turro†‡, and Jingyue Ju*†¶
`
`*Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032; and Departments of †Chemical Engineering,
`‡Chemistry, and §Biomedical Engineering, Columbia University, New York, NY 10027
`
`Contributed by Nicholas J. Turro, March 10, 2005
`
`DNA sequencing by synthesis (SBS) offers an approach for poten-
`tial high-throughput sequencing applications. In this method, the
`ability of an incoming nucleotide to act as a reversible terminator
`for a DNA polymerase reaction is an important requirement to
`unambiguously determine the identity of the incorporated nucle-
`otide before the next nucleotide is added. A free 3ⴕ-OH group on
`the terminal nucleotide of the primer is necessary for the DNA
`polymerase to incorporate an incoming nucleotide. Therefore, if
`the 3ⴕ-OH group of an incoming nucleotide is capped by a chemical
`moiety, it will cause the polymerase reaction to terminate after the
`nucleotide is incorporated into the DNA strand. If the capping
`group is subsequently removed to generate a free 3ⴕ-OH, the
`polymerase reaction will reinitialize. We report here the design and
`synthesis of a 3ⴕ-modified photocleavable fluorescent nucleotide,
`3ⴕ-O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleavable 4,4-
`difluoro-4-bora-3␣,4␣-diaza-s-indacene), as a reversible terminator
`for SBS. This nucleotide analogue contains an allyl moiety capping
`the 3ⴕ-OH group and a fluorophore Bodipy-FL-510 linked to the 5
`position of the uracil through a photocleavable 2-nitrobenzyl
`linker. Here, we have shown that this nucleotide is a good sub-
`strate for a DNA polymerase. After the nucleotide was successfully
`incorporated into a growing DNA strand and the fluorophore was
`photocleaved, the allyl group was removed by using a Pd-catalyzed
`reaction to reinitiate the polymerase reaction, thereby establishing
`the feasibility of using such nucleotide analogues as reversible
`terminators for SBS.
`
`2-nitrobenzyl linker 兩 photocleavage
`
`The completion of the Human Genome Project (1, 2) has led
`
`to an increased demand for high-throughput and rapid DNA
`sequencing methods to identify genetic variants for applications
`in pharmacogenomics (3), disease gene discovery (4, 5), and
`gene function studies (6). Current state-of-the-art DNA se-
`quencing technologies (7–11) to some extent address the accu-
`racy and throughput requirements but suffer limitations with
`respect to cost and data quality. Thus, a new DNA sequencing
`approach is required to broaden the applications of genomic
`information in medical research and health care. In this regard,
`DNA sequencing by synthesis (SBS) offers an alternative ap-
`proach to possibly address the limitations of current DNA
`sequencing techniques. We have previously described the design
`of a parallel chip-based SBS system, which uses a self-priming
`DNA template covalently linked to the glass surface of a chip and
`four modified nucleotides (12–14). The nucleotides are modified
`such that they have a photocleavable fluorescent moiety at-
`tached to the base (5 position of pyrimidines, 7 position of
`purines) and a chemically cleavable group to cap the 3⬘-OH.
`When the correct nucleotide is incorporated in a DNA poly-
`merase reaction, specific to the template sequence, the reaction
`is temporarily terminated because of the lack of a free 3⬘-OH
`group. After the fluorescent signal is detected and the nucleotide
`identified, the 3⬘-OH needs to be regenerated to continue
`incorporating the next nucleotide. In the accompanying report,
`
`we have demonstrated that four photocleavable fluorescent
`nucleotides can be efficiently incorporated by DNA polymerase
`into a growing DNA strand base specifically in a polymerase
`extension reaction, and that the fluorophores can be completely
`removed by photocleavage under near-UV irradiation (⬇355
`nm) with high efficiency (15). Using this system in a four-color
`sequencing assay, we were able to accurately identify multiple
`bases in a self-priming DNA template covalently attached to a
`glass surface.
`Another important requirement for this approach to sequence
`DNA unambiguously is a suitable chemical moiety to cap the
`3⬘-OH of the nucleotide such that it terminates the polymerase
`reaction to allow the identification of the incorporated nucleo-
`tide. The capping group then needs to be efficiently removed to
`regenerate the 3⬘-OH, thereby allowing the polymerase reaction
`to continue. Thus, the photocleavable fluorescent nucleotides
`used in SBS must be reversible terminators of the DNA poly-
`merase reaction to allow the detection of the fluorescent signal
`such that the complementary DNA synthesis and sequence
`identification can be efficiently performed in tandem. The
`principal challenge posed by this requirement is the incorpora-
`tion ability of the 3⬘-modified nucleotide by DNA polymerase
`into the growing DNA strand. The 3⬘ position on the sugar ring
`of a nucleotide is very close to the amino acid residues in the
`active site of the DNA polymerase. This is supported by the 3D
`structure of the previously determined ternary complexes of rat
`DNA polymerase, a DNA template-primer, and dideoxycytidine
`triphosphate (16). Thus, any bulky modification at this position
`provides steric hindrance to the DNA polymerase and prevents
`the nucleotide from being incorporated. A second challenge is
`the efficient removal of the capping group once the fluorescence
`signal is detected. Thus, it is important to use a functional group
`small enough to present no hindrance to DNA polymerase,
`stable enough to withstand DNA extension reaction conditions,
`and able to be removed easily and rapidly to regenerate a free
`3⬘-OH under specific conditions.
`Numerous studies have previously been undertaken to identify
`a 3⬘-modified nucleotide as a substrate for DNA polymerase.
`3⬘-O-methyl nucleotides have been shown to be good substrates
`for several polymerases (17). However, the procedure to chem-
`ically cleave the methyl group is stringent and requires anhydrous
`conditions. Thus, it is not practical to use a methyl group to cap
`the 3⬘-OH group for SBS. It has been reported that nucleotides
`with ether linkages at the 3⬘ position can be incorporated by
`some DNA polymerases, whereas those with ester linkages are
`not generally accepted by most of the polymerases tested (18).
`Significant efforts have been dedicated to evaluating a wide
`
`Abbreviations: PC-Bodipy, photocleavable 4,4-difluoro-4-bora-3␣,4␣,4-diaza-s-indacene;
`SBS, sequencing by synthesis; TPPTS, triphenylphosphinetrisulfonate.
`¶To whom correspondence should be addressed at: Russ Berrie Medical Science Pavilion,
`Columbia Genome Center, Room 405A, Columbia University College of Physicians and
`Surgeons, New York, NY 10032. E-mail: ju@genomecenter.columbia.edu.
`
`© 2005 by The National Academy of Sciences of the USA
`
`5932–5937 兩 PNAS 兩 April 26, 2005 兩 vol. 102 兩 no. 17
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`Scheme 1.
`
`Synthesis of a 3⬘-O-allyl-modified 19-mer oligonucleotide.
`
`oroisopropyl alcohol (pH 8.1). The product was characterized by
`using MALDI-TOF MS.
`
`Deallylation Reaction Performed by Using the 3ⴕ-O-allyl-Modified
`19-mer Oligonucleotide. For the deallylation reaction, we used 55
`eq of Na2PdCl4 and 440 eq of a trisodium triphenylphosphin-
`etrisulfonate (TPPTS) ligand in water at 70°C. Na2PdCl4 in
`degassed water (0.7 ␮l, 2.2 nmol) was added to a solution of
`TPPTS in degassed water (1 ␮l, 17.6 nmol) and mixed well. After
`5 min, a solution of 3⬘-O-allyl-modified oligonucleotide 4 (1 ␮l,
`40 pmol) was added. The reaction mixture was then placed in a
`heating block at 70°C and incubated for 30 sec. The resulting
`deallylated product was desalted by ZipTip (Millipore) and
`analyzed by using MALDI-TOF MS.
`
`Primer Extension Reaction Performed with the Deallylated DNA
`Product. The 10-␮l extension reaction mixture consisted of 45
`pmol of the deallylated DNA product as a primer, 100 pmol of
`a single-stranded synthetic 60-mer DNA template (sequence
`shown in ref. 15) corresponding to a portion of exon 7 of the p53
`gene, 100 pmol of biotin-11–2⬘,3⬘-dideoxyguanosine-5⬘-
`triphosphate (Biotin-11-ddGTP) terminator (PerkinElmer), 1⫻
`Thermo Sequenase reaction buffer, and 4 units of Thermo
`Sequenase DNA polymerase. The extension reaction consisted
`of 15 cycles at 94°C for 20 sec, 48°C for 30 sec, and 60°C for 60
`sec. The product was purified by using solid-phase capture on
`streptavidin-coated magnetic beads (25), desalted by using Zip-
`Tip, and analyzed by using MALDI-TOF MS.
`
`Synthesis of 3ⴕ-O-allyl-dUTP-PC-Bodipy-FL-510. 3⬘-O-allyl-dUTP-
`PC-Bodipy-FL-510 10 was synthesized as shown in Scheme 2.
`Detailed synthesis procedures and characterization data for all
`intermediate compounds (6-9) are described in the supporting
`information.
`PC-Bodipy-FL-510 NHS ester (13) (7.2 mg, 12 mol) in 300 ␮l
`of acetonitrile was added to a solution of 3⬘-O-allyl-5-(3-
`aminoprop-1-ynyl)-2⬘-deoxyuridine-5⬘-triphosphate 9 (2 mg, 4
`mol) in 300 ␮l of Na2CO3-NaHCO3 buffer (0.1 M, pH 8.7). The
`reaction mixture was stirred at room temperature for 3 h. A
`preparative silica-gel TLC plate was used to separate the unre-
`acted PC-Bodipy-FL-510 NHS ester from the fractions contain-
`ing 10 (CHCl3兾CH3OH, 85兾15). The product was concentrated
`further under vacuum and purified with reverse-phase HPLC on
`a 150 ⫻ 4.6-mm C18 column to obtain the pure product 10
`(retention time of 35 min). Mobile phase: A, 8.6 mM triethyl-
`amine兾100 mM hexafluoroisopropyl alcohol in water (pH 8.1);
`B, methanol. Elution was performed with 100% A isocratic over
`10 min, followed by a linear gradient of 0–50% B for 20 min and
`then 50% B isocratic over another 20 min. 3⬘-O-allyl-dUTP-PC-
`
`variety of 3⬘-modified nucleotides to be used as terminators for
`various DNA polymerases and reverse transcriptases, but none
`of the functional groups tested have had established methods to
`regenerate a free 3⬘-OH (19–22).
`It is known that stable chemical functionalities such as allyl
`(OCH2
`OCHACH2) and methoxymethyl (OCH2
`OOOCH3)
`groups can be used to cap an OH group, and can be cleaved
`chemically with high yield (23, 24). We therefore proposed the
`use of such groups as reversible caps for the 3⬘-OH of the
`nucleotide for SBS (12). We report here our efforts in estab-
`lishing the allyl group as a 3⬘-OH capping moiety for the
`nucleotide analogues that can be used in SBS. The choice of this
`group was based on the fact that the allyl moiety, being relatively
`small, would not provide significant hindrance for the polymer-
`ase reaction and would therefore allow the incoming 3⬘-O-allyl-
`modified nucleotide analogue to be accepted by DNA polymer-
`ase. Furthermore, it would be possible to remove this group by
`using catalytic deallylation. Here, we report the design and
`synthesis of a photocleavable fluorescent nucleotide analogue,
`3⬘-O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleav-
`able 4,4-difluoro-4-bora-3␣,4␣-diaza-s-indacene), that can be
`efficiently incorporated by DNA polymerase into a growing
`DNA strand. The allyl group can be rapidly and completely
`removed by a Pd-catalyzed reaction to regenerate a 3⬘-OH
`group, and the deallylated DNA can then allow reinitiation of
`the polymerase reaction to incorporate the subsequent nucleo-
`tide analogue.
`
`Materials and Methods
`All chemicals were purchased from Sigma-Aldrich unless oth-
`erwise indicated. Oligonucleotides used as primers or templates
`were synthesized on an Expedite nucleic acid synthesizer (Ap-
`plied Biosystems). 1H NMR spectra were recorded on a Bruker
`400 spectrometer, and 13C and 31P NMR spectra were recorded
`on a Bruker 300 spectrometer. High-resolution MS data were
`obtained by using a JEOL JMS HX 110A mass spectrometer.
`Mass measurement of DNA was made on a Voyager DE
`MALDI-TOF mass spectrometer (Applied Biosystems). Pho-
`tolysis was performed by using a Spectra Physics GCR-150-30
`Nd-yttrium兾aluminum garnet laser that generates light pulses at
`355 nm (⬇50 mJ per pulse; pulse length of ⬇7 ns) at a frequency
`of 30 Hz with a light intensity at ⬇1.5 W兾cm2. Thermo Seque-
`nase DNA polymerase, HIV-1, and RAV2 reverse transcriptases
`were obtained from Amersham Biosciences. Therminator, Vent
`(exo-), Deep Vent (exo-), Bst, and Klenow (exo-) fragment DNA
`polymerases were obtained from New England Biolabs. 9°N
`polymerase (exo-) A485L兾Y409V was generously provided by
`New England Biolabs. Sequenase V2 DNA polymerase, M-
`MulV, and AMV reverse transcriptases were obtained from
`United States Biochemical. Tfl and Tth DNA polymerases were
`obtained from Promega. Pfu (exo-) DNA polymerase was ob-
`tained from Stratagene. Phosphoramidites and columns for
`nucleic acid synthesis were obtained from Glen Research (Ster-
`ling, VA).
`
`Synthesis of a 3ⴕ-O-allyl-Modified 19-mer Oligonucleotide. 3⬘-O-allyl-
`thymidine phosphoramidite 3, prepared according to Scheme 1
`(also see the supporting information, which is published on the
`PNAS web site), was used to synthesize a 19-mer oligonucleotide,
`5⬘-AGA-GGA-TCC-AAC-CGA-GAC-T(allyl)-3⬘ 4 (molecular
`weight of 5,871). The synthesis was carried out in the 5⬘-to-3⬘
`direction by using 3 along with dA-5⬘-CE, dC-5⬘-CE, dG-5⬘-CE,
`and dT-5⬘-CE phosphoramidites and a dA-5⬘-CPG column. The
`oligonucleotide was purified by HPLC, using an Xterra MS C18
`(4.6 ⫻ 50 mm) column (Waters). The elution was performed
`over 90 min at a flow rate of 0.5 ml兾min and a fixed temperature
`of 50°C, using a linear gradient (12–34.5%) of methanol in a
`buffer containing 8.6 mM triethylamine and 100 mM hexaflu-
`
`Ruparel et al.
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`Scheme 2.
`
`Synthesis of 3⬘-O-allyl-dUTP-PC-Bodipy-FL-510.
`
`Bodipy-FL-510 10 was characterized by the following single-base
`extension reaction and MALDI-TOF MS.
`
`Primer Extension by Using 3ⴕ-O-allyl-dUTP-PC-Bodipy-FL-510 and Pho-
`tocleavage of the Extension Product. An 18-mer oligonucleotide,
`5⬘-AGA-GGA-TCC-AAC-CGA-GAC-3⬘ (molecular weight of
`5,907), was synthesized by using dA-CE, dC-CE, dG-CE, and
`biotin-dT phosphoramidites. A primer extension reaction was
`performed by using a 15-␮l reaction mixture consisting of 50
`pmol of primer, 100 pmol of single-stranded synthetic 60-mer
`DNA template corresponding to a portion of exon 7 of the p53
`gene (15), 200 pmol of 3⬘-O-allyl-dUTP-PC-Bodipy-FL-510, 1⫻
`Thermopol reaction buffer (New England Biolabs), and 15 units
`of 9°N polymerase (exo-) A485L兾Y409V. The extension reaction
`consisted of 15 cycles of 94°C for 20 sec, 48°C for 30 sec, and 60°C
`for 60 sec. A small portion of the DNA extension product 11 was
`desalted by using ZipTip and analyzed by using MALDI-TOF
`MS. The rest of the product was freeze-dried, resuspended in 200
`␮l of deionized water, and irradiated for 10 sec in a quartz cell
`with path lengths of 1.0 cm employing a Nd-yttrium兾aluminum
`garnet laser (⬇355 nm) to cleave the fluorophore from the DNA,
`yielding product 12.
`
`Deallylation of the DNA Extension Product Generated by the Incor-
`poration of 3ⴕ-O-allyl-dUTP-PC-Bodipy-FL-510. The above photo-
`cleaved 3⬘-O-allyl-modified DNA product 12 (180 pmol pro-
`duced in multiple reactions) was dried and resuspended in 1 ␮l
`of deionized H2O. Na2PdCl4 in degassed H2O (4.1 ␮l, 72 nmol)
`was added to a solution of TPPTS in degassed H2O (2.7 ␮l, 9
`nmol) and mixed well. After 5 min, the above DNA product (1
`␮l, 180 pmol) was added. The reaction mixture was then placed
`in a heating block,
`incubated at 70°C for 90 sec to yield
`deallylated product 13, and cooled to room temperature for
`analysis by MALDI-TOF MS.
`
`Polymerase Extension and Photocleavage by Using the Deallylated
`DNA Product as a Primer. The above deallylated DNA product 13
`was used as a primer in a single-base extension reaction. The
`10 ␮l reaction mixture consisted of 50 pmol of the above
`deallylated product 13, 100 pmol of the 60-mer template (15),
`125 pmol of dGTP-PC-Bodipy-FL-510 (14), 4 units of Thermo
`Sequenase DNA polymerase, and 1⫻ reaction buffer. The
`
`extension reaction consisted of 15 cycles of 94°C for 20 sec,
`48°C for 30 sec, and 60°C for 60 sec. The DNA extension
`product 14 was desalted by using the ZipTip protocol, and a
`small portion was analyzed by using MALDI-TOF MS. The
`remaining product was then irradiated with near-UV light for
`10 sec to cleave the fluorophore from the extended DNA
`product. The resulting photocleavage product 15 was desalted
`and analyzed by using MALDI-TOF MS.
`
`Results and Discussion
`In this article, we have shown that an allyl moiety can be
`successfully used as a blocking group for the 3⬘-OH of a
`photocleavable fluorescent nucleotide analogue in SBS to pre-
`vent the DNA polymerase reaction from continuing after the
`incorporation of the 3⬘-O-allyl-modified nucleotide analogue.
`Furthermore, we have demonstrated that the allyl group can be
`efficiently removed to generate a free 3⬘-OH group and allow the
`DNA polymerase reaction to continue to the subsequent cycle.
`Conventional methods for cleavage of the allyl group combine
`a transition metal-catalyzed isomerization of the double bond to
`the enol ether and subsequent hydrolysis of the latter to produce
`the corresponding alcohol (26, 27). For application in SBS, it is
`important to ensure that complete chemical cleavage of the
`3⬘-O-allyl group can be rapidly and specifically carried out while
`leaving the DNA intact. TPPTS has been widely used as a ligand
`for Pd-mediated deallylation under aqueous conditions (28–30),
`whereas an active Pd catalyst can be generated from Na2PdCl4
`and an appropriate ligand (31, 32). Thus, we investigated a
`water-soluble Pd catalyst system generated from Na2PdCl4 and
`TPPTS for deallylation of the 3⬘-O-allyl-modified DNA product.
`To evaluate the cleavage conditions of the allyl group capping
`the 3⬘-OH of DNA, we first synthesized a 19-mer oligonucleotide
`[5⬘-AGAGGATCCAACCGAGAC-T(allyl)-3⬘] using 3⬘-O-allyl-
`thymidine phosphoramidite (Scheme 1). The identity of the
`purified oligonucleotide was established by using MALDI-TOF
`MS. We then tested the above Na2PdCl4兾TPPTS catalyst system
`for the deallylation of the oligonucleotide. In Fig. 1A, the mass
`peak at m兾z 5,871 corresponds to the mass of the purified
`oligonucleotide bearing the allyl group. Fig. 1B shows a single
`mass peak at m兾z 5,831, corresponding to the deallylated DNA
`product, indicating that we were able to achieve near-complete
`deallylation with a DNA兾Na2PdCl4兾TPPTS ratio of 1兾55兾440 in
`
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`
`indicating that the
`corresponding to the extension product,
`deallylated product can be successfully used as a primer in a
`polymerase reaction.
`The above experiments established that Na2PdCl4 and TPPTS
`could be used to efficiently carry out deallylation on DNA in an
`aqueous environment. Our next step was to investigate whether
`a 3⬘-O-allyl-modified nucleotide could be incorporated in a DNA
`polymerase reaction. For this purpose, we synthesized a nucle-
`otide analogue 3⬘-O-allyl-thymidine triphosphate (3⬘-O-allyl-
`dTTP) (supporting information), which was tested with 15
`different polymerases for incorporation. The tested enzymes
`included Therminator, Thermo Sequenase, Vent (exo-), Deep
`Vent (exo-), Tth, Tfl, Bst, Pfu (exo-), Klenow (exo-) fragment
`and Sequenase DNA polymerases, AMV, RAV2, M-MulV, HIV
`reverse transcriptases, and a 9°N polymerase (exo-) bearing the
`mutations A485L and Y409V. Our preliminary results showed
`that 9°N DNA polymerase (exo-) A485L兾Y409V could effi-
`ciently incorporate 3⬘-O-allyl-dTTP in an extension reaction,
`consistent with results reported recently (31).
`After confirming the incorporation ability of 3⬘-O-allyl-dTTP
`into a growing DNA strand by DNA polymerase, we then
`synthesized a new 3⬘-modified photocleavable fluorescent nu-
`cleotide analogue, 3⬘-O-allyl-dUTP-PC-Bodipy-FL-510, accord-
`ing to Scheme 2, and established that it also can be efficiently
`incorporated by the above polymerase. The aim was to evaluate
`that the presence of the bulky photocleavable fluorescent moiety
`on the base and the allyl group on the 3⬘ end of the nucleotide
`analogue would not affect the polymerase extension reaction.
`Furthermore, we wanted to demonstrate an entire cycle of
`primer extension, photocleavage of the fluorophore, deallylation
`followed by extension with another photocleavable fluorescent
`nucleotide complementary to the next base on the template, and
`photocleavage once again. This experiment will thus test the
`feasibility of using 3⬘-O-allyl-dUTP-PC-Bodipy-FL-510 as a re-
`versible terminator for SBS.
`The entire cycle of a polymerase reaction using 3⬘-O-allyl-
`dUTP-PC-Bodipy-FL-510 as a reversible terminator is depicted
`in Scheme 3. The extension product 11 obtained by using
`3⬘-O-allyl-dUTP-PC-Bodipy-FL-510 and 9°N DNA Polymerase
`(exo-) A485L兾Y409V was purified by using HPLC and analyzed
`by using MALDI-TOF MS. The base in the template immedi-
`ately adjacent to the priming site was ‘‘A.’’ Thus, if 3⬘-O-allyl-
`dUTP-PC-Bodipy-FL-510 was accepted by the polymerase as a
`terminator, the primer would extend by one base and then the
`reaction would terminate. Our results indicate that this was
`indeed the case. After confirming that the extension reaction was
`
`Schematic representation (Left) and step-by-step MALDI-TOF MS
`Fig. 1.
`results (Right) for the deallylation of a 3⬘-O-allyl-modified oligonucleotide
`and the use of the deallylated oligonucleotide as a primer in a polymerase
`extension reaction. (A) Peak at m兾z 5,871 corresponding to the HPLC-purified
`3⬘-O-allyl-modified 19-mer oligonucleotide. (B) Peak at m兾z 5,831 correspond-
`ing to the above oligonucleotide without the allyl group, obtained after 30 sec
`of incubation with Na2PdCl4 and TPPTS [P(PhSO3Na)3] at 70°C. (C) Peak at m兾z
`6,535 corresponding to the extension of the deallylated oligonucleotide by
`Biotin-11-ddGTP using Thermo Sequenase DNA polymerase.
`
`a reaction time of 30 sec. The next step was to prove that the
`above deallylated DNA product could be used as a primer in a
`polymerase extension reaction. We therefore carried out a
`single-base extension reaction using the deallylated DNA prod-
`uct as a primer, a synthetic template, and a biotin-11-ddGTP
`nucleotide terminator that was complementary to the base
`immediately adjacent to the priming site on the template. The
`DNA extension product was isolated by using solid-phase cap-
`ture purification and analyzed by using MALDI-TOF MS (25).
`The mass spectrum in Fig. 1C shows a clear peak at m兾z 6,535
`
`Scheme 3. One entire polymerase reaction cycle using 3⬘-O-allyl-dUTP-PC-Bodipy-FL-510 as a reversible terminator.
`
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`successful, we irradiated it with near-UV light at 355 nm for 10
`sec to cleave the fluorophore from the DNA, generating product
`12. In an SBS system, this step would ensure that there would be
`no carryover of the fluorescence signal into the next incorpo-
`ration cycle so as to prevent the generation of ambiguous data
`at each step, as shown in the companion article (15). The
`photocleavage product 12 was then incubated with a Na2PdCl4兾
`TPPTS catalyst system at 70°C for 90 sec to perform deallylation.
`The deallylated DNA product 13 was purified by reverse-phase
`HPLC and then used as a primer in a second DNA extension
`reaction to prove that the regenerated 3⬘-OH was capable of
`allowing the polymerase reaction to continue. For the extension
`reaction, we used a photocleavable fluorescent nucleotide
`dGTP-PC-Bodipy-FL-510 and Thermo Sequenase DNA poly-
`merase. The extension product 14 was irradiated as above for 10
`sec to generate photocleavage product 15 and hence complete an
`entire reversible termination cycle.
`After each step in the above cycle, a portion of the product was
`purified and analyzed by using MALDI-TOF MS to confirm its
`identity and the successful completion of that step. Each product
`was desalted by using the ZipTip desalting protocol to ensure the
`generation of sharp and well resolved data free from salt peaks.
`The MALDI-TOF MS data for the product in each step are
`shown in Fig. 2. Fig. 2 A shows the primer extension product 11
`at m兾z 6,787 generated by using 3⬘-O-allyl-dUTP-PC-Bodipy-
`FL-510. The peak at m兾z 6,292 corresponds to the photocleavage
`product that was generated by the partial photocleavage of the
`extension product due to the nitrogen laser (⬇337 nm) used for
`ionization of the analyte in MALDI-TOF MS. Fig. 2B shows the
`photocleavage result after the 10-sec irradiation of the extension
`product at 355 nm. It can be seen from the data that the peak
`at m兾z 6,787, corresponding to the extension product, has
`completely vanished, and only a single peak corresponding to 12
`remains at m兾z 6,292, which proves that photocleavage was
`efficiently achieved. Fig. 2C shows a similar single peak at m兾z
`6,252, which corresponds to the deallylated photocleavage prod-
`uct 13. The absence of a significant peak at m兾z 6,292 proves that
`deallylation was completed with high efficiency. Fig. 2D shows
`the MALDI-TOF MS data for the extension product obtained
`by using the above deallylated DNA product 13 as a primer and
`nucleotide analogue dGTP-PC-Bodipy-FL-510. A dominant
`peak is seen at m兾z 7,133 corresponding to the extension product
`14. Finally, Fig. 2E shows a clear peak at m兾z 6,637 correspond-
`ing to the photocleavage product 15 and no significant peak at
`m兾z 7,133, indicating that complete photocleavage had occurred.
`The results of the above experiments provide sufficient proof of
`the feasibility of using the allyl group as a reversible capping moiety
`for the 3⬘-OH of the photocleavable nucleotide analogues for SBS,
`validating the approach we had previously proposed (12). We have
`shown that a 3⬘-O-allyl-modified nucleotide bearing a photocleav-
`able fluorophore is an excellent substrate for 9°N DNA polymerase
`A485L兾Y409V and can be incorporated with high efficiency in a
`polymerase extension reaction. We have also demonstrated that
`complete photocleavage is achieved in ⬇10 sec on these DNA
`products. Furthermore, we have shown that deallylation can be
`swiftly achieved to near completion under mild reaction conditions
`in an aqueous environment by using a palladium catalyst. Finally,
`we have established that the deallylated DNA product can be used
`as a primer to continue the polymerase reaction and that extension
`and photocleavage can be performed with high efficiency. These
`findings confirm that an allyl moiety protecting the 3⬘-OH group
`indeed bestows the capability of reversible terminating abilities to
`photocleavable nucleotide analogues, which can be used for SBS to
`facilitate the development of this approach for high-throughput
`DNA sequencing and genotyping.
`
`We thank New England Biolabs for generously providing the 9°N DNA
`polymerase (exo-) A485L兾Y409V. This work was supported by National
`
`Fig. 2. MALDI-TOF MS results for each step of a polymerase reaction cycle
`using 3⬘-O-allyl-dUTP-PC-Bodipy-FL-510 as a reversible terminator. (A) Peak at
`m兾z 6,787 corresponding to the primer extension product 11 obtained by
`using 3⬘-O-allyl-dUTP-PC-Bodipy-FL-510 and the 9°N polymerase (exo-) A485L兾
`Y409V. (B) Peak at m兾z 6,292 corresponding to the photocleavage product 12.
`(C) Peak at m兾z 6,252 corresponding to the photocleavage product without
`the allyl group 13 obtained after 90 sec of incubation with the catalyst and
`ligand at 70°C. (D) Peak at m兾z 7,133 corresponding to the extension product
`14 from the purified deallylated product using dGTP-PC-Bodipy-FL-510 and
`Thermo Sequenase DNA polymerase. (E) Peak at m兾z 6,637 corresponding to
`the photocleavage product 15.
`
`Institutes of Health Center of Excellence in Genomic Science Grants P50
`HG002806 and R01 HG003582 and the Packard Fellowship for Science
`and Engineering.
`
`5936 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0501962102
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`Ruparel et al.
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