The fluoride cleavable 2-(cyanoethoxy)methyl (CEM) group as reversible 3′-O-terminator for DNA sequencing-by-synthesis - synthesis, incorporation, and cleavage

Article abstract of DOI:10.1080/15257770701257301

A new and promising sequencing technology called sequencing-by-synthesis (SBS) enables fast determination of DNA sequences. 2′-Deoxynucleotides containing the (2-cyanoethoxy)methyl (CEM) group at the 3′-O-position are potential reversible terminators for

Full text of DOI:10.1080/15257770701257301

Nucleosides, Nucleotides, and Nucleic Acids, 26:271–275, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701257301
THE FLUORIDE CLEAVABLE 2-(CYANOETHOXY)METHYL (CEM)
GROUP AS REVERSIBLE 3ꢀ-O-TERMINATOR FOR DNA
SEQUENCING-BY-SYNTHESIS—SYNTHESIS, INCORPORATION, AND
CLEAVAGE
A. Fo¨ ldesi
A. Keller
Quiatech AB, Uppsala, Sweden
2
Institut fu¨r Organische Chemie und Chemische Biologie, J.W. Goethe
2
Universita¨t Frankfurt, Frankfurt, Germany
A. Stura and S. Zigmantas Fermentas UAB, Vilnius, Lithuania
M. Kwiatkowski Quiatech AB (now Oligovation), Uppsala, Sweden
D. Knapp and J. W. Engels Institut fu¨r Organische Chemie und Chemische
2
2
2
Biologie, J.W. Goethe Universita¨t Frankfurt, Frankfurt, Germany
A new and promising sequencing technology called sequencing-by-synthesis (SBS) enables fast
2
determination of DNA sequences. 2 ꢁ-Deoxynucleotides containing the (2-cyanoethoxy)methyl (CEM)
group at the 3 ꢁ-O-position are potential reversible terminators for the SBS technology. Herein we
describe the synthesis, the incorporation by several polymerases, and the cleavage of this 3 ꢁ-O-
blocking group using 3 ꢁ-O-CEM-thymidinyl-5 ꢁ-O-triphosphate 7 as an example.
Keywords DNA sequencing-by-synthesis; reversible terminator; triphosphate synthesis;
3ꢁ-O-alkylation
INTRODUCTION
Today’s methods for DNA sequencing are mainly limited by the use of
gel electrophoretic separation of the extension products.[1] Future demands
for understanding, diagnostics, treatment, and prevention of diseases
however create a need for faster and more cost effective sequencing
methods. The idea of cyclic-array sequencing-by-synthesis (SBS) has the
Received 20 September 2006; accepted 6 December 2006.
We would like to thank the EU FP6 CRAFT project “ArraySBS” 017623 for financial support.
Address correspondence to J. W. Engels, Institut fu¨r Organische Chemie und Chemische Biologie,
J. W. Goethe Universita¨t Frankfurt a. M., Max-von-Laue Str. 7, D-60438, Frankfurt a. M., Germany. E-mail:
joachim.engels@chemie.uni-frankfurt.de
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potential to fulfil these needs. This method is based on incorporation
and immediate identification of every single nucleotide; hence, it is not
based on gel electrophoresis. There are already some systems that are
based on SBS-technologies such as fluorescent in situ sequencing[2] and
pyrosequencing.[3] Drawbacks of these methods are that they need an
amplification of the DNA to be sequenced and that they can be inaccurate
concerning the length of homopolymeric sequences. A new and promising
approach for the realisation of the SBS-technology without the above men-
tioned disadvantages is the use of 3ꢁ-O-modified, reversibly terminating, dye
labelled nucleotides.[4,5] Here the polymerase reaction is stopped after each
incorporation step and the nucleotide can be identified by the spectrum of
the attached fluorescent dye. After identification the 3ꢁ-O-modification is
cleaved and the next cycle starts. The ultimate object of this approach is the
direct sequencing of single molecules of DNA arranged on an array to avoid
the amplification step.
A protecting group for the 3ꢁ-O-position is suitable for SBS if it effec-
tively terminates the polymerase reaction and can be removed fast and
quantitative afterward. We turned our attention to a publication describing
the novel (2-cyanoethoxy)methyl (CEM) 2ꢁ-O-protecting group for RNA
synthesis.[6] They found that this group is readily removable upon treatment
with tetrabutylammonium fluoride (TBAF) in dry tetrahydrofuran (THF).
We asked the question if the mentioned group could be used for protection
of the 3ꢁ-O-position in deoxynucleotides, and further, if the deprotection un-
der such nonbiological conditions is possible without affecting the duplex
structure of the DNA-template. After performing a series of experiments
described herein we found the answer to be positive.
Synthesis of 3ꢁ-O-(2-Cyanoethoxy)methyl-thymidinyl-5ꢁ-O-
triphosphate 7
The synthesis of the new 3ꢁ-O-modified nucleotide 7 is shown in
Scheme 1. The formacetal moiety of the modification was introduced
by using the 3ꢁ-O-methylthiomethyl substituted nucleoside 3.[7] This was
converted into its reactive chloro-derivative 4 which then reacted in situ
with 3-hydroxypropionitrile to form the CEM group at the 3ꢁ-O-position of
nucleoside 5. The corresponding triphosphate 7 was synthesised according
to the Ludwig-Eckstein procedure[8] and after ion exchange FPLC and
RP-HPLC the pure triphosphate 7 was obtained.
Incorporation Tests
For the incorporation tests different commercially available DNA poly-
merases and thermostable polymerases modified by Fermentas UAB were

CEM Group as Reversible 3ꢁ-O-Terminator for DNA Sequencing-by-Synthesis
273
S◦CHEME 1 Synthesis of 3ꢁ-O-CEM-thymidinyl-5ꢁ-O-triphosphate 7. i) BzCl, dry pyridine, 3.5 hours, at
˚
0 C, 87%; ii) DMSO, AcOH, Ac2O, 25 hours at room temperature, 81%; iii) a) Et3N, 3 A MS, dry DCE,
2 hours at room temperature b) SO2Cl2, 1 hour at 0◦C; iv) 3-hydroxypropionitrile, 10 minutes at 0◦C,
2 hours at room temperature, 55%; v) MeOH, 32% aq. NH3, 5 hours at room temperature; vi) a) 1 M
2-chloro-4H-1,2,3-benzodioxaphosphorin-4-one in dry 1,4-dioxane, dry pyridine, dry DMF, 20 minutes at
room temperature b) 0.5 M (n-Bu3N)2H2P2O7, dry DMF, n-Bu3N, 45 minutes at room temperature c)
1% I2 in pyridine/H2O 98:2, 30 minutes at room temperature d) 32% aq. NH3, 15%.
used. They were incubated for 60 minutes at 37◦C (buffer Tango) with
50 µM 3ꢁ-O-CEM-dTTP and 10 nM of the radioactively (33P) labelled DNA
duplex:
5ꢁ-33P-TGCAGGCATGCAAGCTTGGCGTA
3ꢁ- ACGTCCGATCGTTCGAACCGCATAAAAAAAAAAAA
After 60 minutes of incubation, 100 µM dTTP were added and the
reaction was allowed to proceed for additional 15 minutes at 37◦C.
Figure 1 shows the results of the polymerase screening. The best
results of incorporation and termination were achieved with pol6ꢁꢁ. The

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A. Fo¨ldesi et al.
FIGURE 1 Polyacrylamid gel of the incorporation of 3ꢁ-O-CEM modified dTTP 7 by several polymerases,
detection was carried through the radioactively (33P) labelled DNA duplex.
modified nucleotide 7 is accepted very well and shows efficient terminating
properties.
Cleavage Experiments
Cleavage experiments were performed using a 24-mer oligonucleotide
that contains the 3ꢁ-O-modified thymidine at the 3ꢁ-end (5ꢁ-TGCAGGC
ATGCAAGCTTGGCGTATCEM-3ꢁ). The 3ꢁ-O-modified thymidine was in-
corporated by the corresponding 5ꢁ-O-phosphoramidite made in our
FIGURE 2 Analysis of the cleavage experiments of 3ꢁ-O-CEM with TBAF in dry THF by RP-HPLC
(Gemini C18, Phenomenex HPLC column), buffers: A: 5% ACN in 0.1 M TEAAc, B: 80% ACN in 0.1 M
TEAAc, gradient: 0–40% B in 12 minutes.

CEM Group as Reversible 3ꢁ-O-Terminator for DNA Sequencing-by-Synthesis
275
laboratory. The deprotected and HPLC purified material was treated with
TBAF under the specific conditions presented for every RP-HPLC chro-
matogram in Figure 2. The analysis was performed on a Gemini C18,
Phenomenex HPLC column using buffer A: 5% ACN in 0.1 M TEAAc and
buffer B: 80% ACN in 0.1 M TEAAc, gradient: 0–40% B in 12 minutes.
The results in Figure 2 show that the cleavage proceeded very fast when
treating the oligonucleotides with 1.3 µmol TBAF (1 M solution in dry
THF) per pmol CEM (5 minutes at room temperature, 2 minutes at 37◦C),
furthermore no side product formation was observed.
Outlook
With this new 3ꢁ-O-CEM modified nucleotide 7 a proof of principle for
the suitability of this protecting group as a reversible terminator could be
achieved. A polymerase was identified that accepts the 3ꢁ-O-modification
very well and efficient termination could be observed. The cleavage under
the expected conditions also proceeded very quickly and quantitatively.
At the moment the syntheses and testing of the other three modified
nucleotides as well as the labelling with fluorescent dyes are in progress.
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