Template-directed synthesis in 3′- and 5′-direction with reversible termination

Article abstract of DOI:10.1002/anie.201203859

Extending both ways: A method for DNA-templated synthesis on solid support is described. Controlled, stepwise chain extension was demonstrated both in the direction favored by nature (3'-extension; see scheme) and in the direction typical for conventional DNA synthesizers (5'-extension). Copyright

Full text of DOI:10.1002/anie.201203859

Angewandte
Chemie
DOI: 10.1002/anie.201203859
DNA-Directed Synthesis
Template-Directed Synthesis in 3’- and 5’-Direction with Reversible
Termination**
Andreas Kaiser, Sebastian Spies, Tanja Lommel, and Clemens Richert*
Template-directed synthesis of nucleic acid chains is one of
the most important processes of the animate world. In the
cell, polymerases catalyze the extension of primers bound to
DNA templates, using nucleoside 5’-triphosphates as mono-
mers. Chain growth occurs in 3’-direction.[1] Chemists, using
programmable DNA synthesizers, commonly perform DNA
and RNA syntheses in template-free fashion through the
coupling of 3’-phosphoramidites of protected nucleosides,
with chains growing in 5’-direction on a solid support.[2]
Control over the sequence and length of the synthetic
oligonucleotides is achieved through protecting groups and
the programmed flow of solutions.
we refer to as chemical primer extension with reversible
termination. This method uses protecting groups that are
removable under non-denaturing conditions and a solid
support on which both template and primer are bound
noncovalently. Controlled, stepwise synthesis of oligonucle-
otides in template-directed fashion is demonstrated both in 3’-
and 5’-direction.
Chemical primer extension (CPE) is the template-
directed elongation of an oligonucleotide primer through
the reaction of activated nucleotides with the primer terminus
in the absence of enzymes. Chemical primer extension assays
have been performed for the elongation of RNA,[3–5] DNA,[6]
3’-amino-terminal DNA,[7–9] 2’-amino-terminal DNA,[10,11]
and more-extensively backbone-modified nucleic acids.[12]
With CPE, the intrinsic ability of nucleic acids to copy and
possibly replicate genetic information can be studied.[13] Most
studies, including some on template-free polymerization of
nucleotides[14,15] and short oligonucleotides,[16] have focused
on prebiotic questions. An increasing number of theoretical
studies are appearing.[17–19] But, recent work has also demon-
strated the potential of CPE[8a,20] and chemically induced
ligation[21] for practical applications. Multiple, stepwise chain
extensions, controlled by protecting groups are currently
unknown.[22] Also, to the best of our knowledge, enzyme-free
primer extension has thus far been limited to chain growth in
3’/2’-direction.
As part of a program on enzyme-free sequence determi-
nation by CPE, we have previously shown that azaoxybenzo-
triazole esters (OAt esters) give faster extensions than
methylimidazolides.[8a,c] Near quantitative CPE was observed
for 64 different sequence contexts with OAt esters of
deoxynucleotides.[8c] Herein, we present a new method that
[*] Dipl.-Chem. A. Kaiser, Dipl.-Chem. S. Spies, Prof. C. Richert
Institut fꢀr Organische Chemie, Universitꢁt Stuttgart
70569 Stuttgart (Germany)
E-mail: lehrstuhl-2@oc.uni-stuttgart.de
Dr. T. Lommel
Scheme 1. Extension reactions and method for template-directed syn-
thesis with reversible termination. a) Primer extension in 3’-direction,
giving N3’!P5’ phosphoramidate DNA. b) Extension in 5’-direction,
giving P3’!N5’ phosphoramidate DNA. c) Solid supports 3 and 4 for
primer extension in 3’-direction, and d) support 5 for primer extension
in 5’-direction. Full sequences of templates and capture oligonucleo-
tides are given in the Supporting Information. All strands in this study
are oligodeoxynucleotides. Azoc=CO-OCH2N3.
Product Development, Research and Quality
Thyssen Krupp Rasselstein AG, 56626 Andernach (Germany)
[**] This work was supported by the DFG (grant no. RI 1063/8-1 to C.R.)
The authors thank Dr. E. Kervio and R. Haug for helpful discussions,
and D. Gçhringer, and T. Sabirov for skilled technical assistance.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201203859.
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We chose genetic polymers with phosphoramidate link-
ages that are isoelectronic to natural DNA for our study
(Schemes 2a and 2b).[7–11,16,21–23] The azidomethyloxycarbonyl
(Azoc) group, first described by Pothukanuri and Wins-
singer,[24] was selected for the protection of the 3’- and 5’-
amino group of the mononucleotides (1a–t, 2c, and 2t;
Schemes 1a and 1b) and was introduced using a recently
developed carbonate reagent.[25]
The syntheses of Azoc-protected monomers 1a, 1c, 1g, 1t,
2c, and 2t are shown in the Supporting Information. Amino-
terminal oligodeoxynucleotide primers were synthesized
using literature protocols.[8c,26] The Azoc protection chosen
avoids uncontrolled polymerization and intramolecular cycli-
zation,[11a] and deprotection of the 3’-, or 5’-amino group after
the elongation can be induced with a water-soluble phos-
phine. High yields are required for both the extension and the
deprotection reaction for efficient, multiple primer extension.
Azidomethyl groups have been employed in second gener-
ation sequencing to protect 3’-hydroxy groups reversibly.[27,28]
These are not suitable for amines. The Azoc group is smaller
than common protecting groups for amines,[29] thus avoiding
steric hindrance during monomer binding. The Azoc group
performed better than larger, photolabile protecting groups,
which were tested during the early stages of our study.[8b,22]
To establish a successful protocol for primer extension in
a controlled, stepwise fashion, with cleavage of the protecting
groups under non-denaturing conditions, we tested several
solid supports. First, streptavidin-coated magnetic beads and
biotin-labeled capture oligonucleotides were tested, a combi-
nation described earlier for RNA-templated reactions and
twofold primer extension with 3’-aminoprimers.[5c,8b] The
protein-coated surface proved reactive towards 3’-amino-
primers, as indicated by signal loss in MALDI-TOF mass
spectra and the results of control experiments. Switching to
epoxy-modified magnetic beads, combined with amino- or
thiol-modified capture oligonucleotides gave magnetic-bead-
based supports 3, 4, and 5, which do not suffer from this
limitation. Supports 3 and 4 were used for CPE in 3’-direction
(Scheme 1c) and 5 was used for CPE in 5’-direction
(Scheme 1d). The Supporting Information provides details
and characterization data for both types of DNA-loaded
magnetic beads. The active form of the supports was
generated by hybridizing template and primer to the capture
oligonucleotide on their surface.
With monomers and supports in hand, we performed
template-directed syntheses (Scheme 2). First, we tested
Azoc-protected 1t as the monomer, and tris(2-carboxyethyl)-
phosphine (TCEP) in aqueous buffer as the cleavage reagent
in the deprotection step that involves a Staudinger reduction.
High yields in extension and deprotection were observed for
ten cycles, as demonstrated by MALDI-TOF MS readout
after every other elongation cycle (Scheme 2a).
Scheme 2. Tenfold template-directed synthesis with reversible termination. a) Extension of aminoprimer 7 on poly(dA) template 6 in 3’-direction,
using thymidine 1t (100 mm) and pyridine (50 mm)[8b] at 48C for 6 h. Deprotection: TCEP (250 mm) at 208C for 10 min; b) Extension of
aminoprimer 10 on mixed template 9 in 3’-direction, using monomers 1a, 1c, 1g, or 1t (40 mm of the complementary monomer) at 208C for 4–
12 h; Deprotection: TCEP (100 mm) at 08C for 30 min; c) Extension of aminoprimer 13 on GA-template 12 in 5’-direction, using monomers 2c or
2t under conditions otherwise identical to those given for b). The upper part of each graphic shows the reaction scheme and the lower part
shows overlays of MALDI-TOF MS spectra acquired during the assay. All extension steps were performed in buffer containing HEPBS (200 mm),
NaCl (400 mm), and MgCl2 (80 mm) at pH 8.9. All deprotection steps used the same buffer additionally containing TCEP (100 or 250 mm, as
detailed above). Primer sections with phosphoramidate linkages are shown in italics. HEPBS=N-(2-hydroxyethyl)piperazine-N’-(4-butanesulfonic
acid).
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Template-directed synthesis in the opposite (5’) direction
was also first tested with a thymidine monomer (2t). Again,
mass spectra were obtained from small samples of the support
that were denatured by heating in deionized water (see the
Experimental Section for details). After eight extension
cycles, the extended primer was still detectable by MALDI-
TOF MS, before the signal disappeared in the noise. Our
study demonstrates, for the first time, that CPE in 5’-direction
is of a similar efficiency as the established primer extension in
3’-direction.
primer was observed, with little singly extended primer
remaining when the assay was stopped (Scheme 3b).
In conclusion, we have developed protected monomers
and solid supports for chemical primer extension in 3’- and 5’-
direction with reversible termination. To the best of our
knowledge, our results include the first instance that CPE was
shown to be an efficient process for extension both in 3’- and
We then studied extension on mixed-sequence templates.
The tenfold primer extension in 3’-direction was observed on
two different template sequences containing all four different
nucleobases (Scheme 2b and Supporting Information). Fur-
thermore, tenfold extension of the 5’-amino-terminal primer
was induced by performing extension cycles with 2c or 2t,
using a template with A and G as templating bases
(Scheme 2c).
Since the MALDI-TOF MS signal becomes increasingly
weak as the primer grows, the completeness of a CPE reaction
with reversible termination was checked by performing an
assay with a Cy3-labeled 3’-aminoprimer and analyzing the
product by polyacrylamide gel electrophoreses after tenfold
extension (see the Supporting Information, Scheme S11).
Finally, exploratory experiments were performed with
mixtures of nucleotide monomers to test the sequence fidelity
of the CPE. Determining fidelity is important for a better
understanding of the ability of genetic polymers to undergo
spontaneous replication under potentially prebiotic condi-
tions.[13b] It is also important for possible applications, such as
enzyme-free sequencing. The ratio of monomers chosen was
5:2:2:1 (1t/1a/1c/1g), a mixture that roughly reflects the
relative incorporation propensity of the nucleotides to
undergo incorporation.[9b] We performed fourfold CPE with
reversible termination using three different templates and
found that in all cases the most prominent peak corresponded
to the mass of the correctly extended primer (Scheme 3 and
the Supporting Information). A more detailed study is under
way and will be reported separately.
A first experiment using a Janus-headed primer, with
amino groups at both the 3’- and the 5’-terminus was
performed, using a system where the template strand was
immobilized directly on the solid support. When this doubly
reactive primer was exposed to a mixture of the required 3’-
reactive and 5’-reactive monomers, the correctly extended
Scheme 3. a) Fourfold template-directed synthesis in 3’-direction with
reversible termination. Extension of aminoprimer 10 on mixed tem-
plate 9 using a mixture of monomers 1t (40 mm), 1a (16 mm) 1c
(16 mm), and 1g (8 mm) at 208C for 12 h. b) Simultaneous template-
directed synthesis in 3’- and 5’-direction. Extension of aminoprimer 17
on immobilized template 16 using a mixture of monomers 2c
(40 mm), and 1a (10 mm) at 48C for 12 h. Immobilization of the
template to give solid support 16 was performed using the same
protocol as for solid support 5. All extension steps were performed in
buffer containing HEPBS (200 mm), NaCl (400 mm), and MgCl2
(80 mm) at pH 8.9. All deprotection steps were performed at 08C for
30 min and used the same buffer additionally containing TCEP
(100 mm). Primer sections with phosphoramidate linkages are shown
in italics.
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845; b) M. Hey, C. Hartel, M. W. Gçbel, Helv. Chim. Acta 2003,
86, 844 – 854.
[5] a) S. R. Vogel, C. Deck, C. Richert, Chem. Commun. 2005,
4922 – 4924; b) S. R. Vogel, C. Richert, Chem. Commun. 2007,
1896 – 1898; c) C. Deck, M. Jauker, C. Richert, Nat. Chem. 2011,
3, 603 – 608.
5’-direction. The exquisite control over individual steps
through the Azoc protecting group allows for the determi-
nation of sequence composition after each step. Our results
also establish CPE as a third method for the synthesis of
genetic polymers. The method complements nontemplated
chemical synthesis and template-directed polymerase-cata-
lyzed synthesis. It has not escaped our attention that CPE with
reversible termination may be extended to nonenzymatic
replication systems that reveal the intrinsic sequence prefer-
ence of enzyme-free and ribozyme-free, nucleotide-based
replication systems and possibly leads to new functional
entities through selections that do not require enzymes. We
are actively pursuing the experimental realization of such
systems.
[6] F. Olasagasti, H. J. Kim, N. Pourmand, D. W. Deamer, Biochimie
2011, 93, 556 – 561.
[7] R. Lohrmann, L. E. Orgel, J. Mol. Biol. 1977, 109, 323 – 328.
[8] a) P. Hagenbuch, E. Kervio, A. Hochgesand, U. Plutowski, C.
Richert, Angew. Chem. 2005, 117, 6746 – 6750; Angew. Chem.
Int. Ed. 2005, 44, 6588 – 6592; b) M. Rçthlingshçfer, E. Kervio, T.
Lommel, U. Plutowski, A. Hochgesand, C. Richert, Angew.
Chem. 2008, 120, 6154 – 6157; Angew. Chem. Int. Ed. 2008, 47,
6065 – 6068; c) E. Kervio, A. Hochgesand, U. Steiner, C. Richert,
Proc. Natl. Acad. Sci. USA 2010, 107, 12074 – 12079; d) M.
Rçthlingshçfer, C. Richert, J. Org. Chem. 2010, 75, 3945 – 3952.
[9] a) S. Rajamani, J. K. Ichida, T. Antal, D. A. Treco, K. Leu, M. A.
Nowak, J. W. Szostak, I. A. Chen, J. Am. Chem. Soc. 2010, 132,
5880 – 5885; b) K. Leu, B. Obermayer, S. Rajamani, U. Gerland,
I. A. Chen, Nucleic Acids Res. 2011, 39, 8135 – 8147.
[10] R. Lohrmann, L. E. Orgel, Nature 1976, 261, 342 – 344.
[11] a) S. S. Mansy, J. P. Schrum, M. Krishnamurthy, S. Tobe, D. A.
Treco, J. W. Szostak, Nature 2008, 454, 122 – 125; b) J. P. Schrum,
A. Ricardo, M. Krishnamurthy, J. C. Blain, J. W. Szostak, J. Am.
Chem. Soc. 2009, 131, 14560 – 14570.
[12] a) J. J. Chen, X. Cai, J. W. Szostak, J. Am. Chem. Soc. 2009, 131,
2119 – 2121; b) N. M. Bell, R. Wong, J. Micklefield, Chem. Eur. J.
2010, 16, 2026 – 2030; c) B. D. Heuberger, C. Switzer, Org. Lett.
2006, 8, 5809 – 5811; d) I. A. Kozlov, P. K. Politis, A. Van Aer-
schot, R. Busson, P. Herdewijn, L. E. Orgel, J. Am. Chem. Soc.
1999, 121, 2653 – 2656; e) I. A. Kozlov, M. Zielinski, B. Allart, L.
Kerremans, A. Van Aerschot, R. Busson, P. Herdewijn, L. E.
Orgel, Chem. Eur. J. 2000, 6, 151 – 155; f) X. Li, Z.-Y. J. Zhan, R.
Knipe, D. G. Lynn, J. Am. Chem. Soc. 2002, 124, 746 – 747.
[13] a) J. W. Szostak, D. B. Bartel, P. L. Luisi, Nature 2001, 409, 387 –
390; b) J. W. Szostak, J. Syst. Chem. 2012, 3, 2.
Experimental Section
General protocol for CPE assays. A slurry of magnetic beads with
immobilized capture oligonucleotide (25 mL, 10 mgmLꢀ1, capacity
approx. 200 pmolmgꢀ1
; 50 pmol oligonucleotide) was added to
a polypropylene vessel. The storage buffer (PBS buffer containing
0.02% NaN3) was removed using magnetic separation. The beads
were washed with washing buffer (2 ꢀ 5 mL; HEPBS (200 mm), NaCl
(400 mm), MgCl2 (80 mm) ; pH 8.9). A stock solution of HEPBS buffer
(2 mL; HEPBS (500 mm), NaCl (1m), MgCl2 (200 mm) ; pH 8.9),
solutions of template (0.75 mL, 75 pmol), 3’-aminoprimer (0.75 mL,
75 pmol), and water (1.5 mL) were added, to give a final volume of
5 mL. Annealing was induced by cooling from 808C (5 min) to 408C
(1 h) and then to 208C, with a cooling rate of 0.18Csꢀ1 between the
plateaus. After magnetic separation, beads were washed with washing
buffer (2 ꢀ 2 mL), Then, stock solution of HEPBS buffer (2 mL) and
a solution of the monomer (1a, 1c, 1g, or 1t; 3 mL, 66.6 mm) were
added, leading to the desired concentrations of oligonucleotides
(10 mm), HEPBS (200 mm), NaCl (400 mm), MgCl2 (80 mm), and 1a,
1c, 1g, or 1t (40 mm). After vortexing, CPE was allowed to proceed at
208C for 4 h (1a, 1c, and 1g) or 12 h (1t). The beads were washed
with stock solution of the HEPBS buffer (2 ꢀ 2 mL). Then, the stock
solution of HEPBS buffer (2 mL), water (2 mL), and a solution of
TCEP (0.5m, 1 mL) was added, and deprotection was allowed to
proceed at 08C for 30 min. For monitoring, a sample was drawn
(0.15 mL) and washed with ammonium acetate solution (3 ꢀ 1 mL, 1m;
pH 7). Water (5 mL) was added, and the resulting suspension was
heated to 808C. The hot supernatant was immediately aspirated,
using a magnet to retain the beads, and added to an aqueous
suspension of Dowex 50 WX8-200 cation exchange beads (1 mL,
ammonium form) for 25 min. An aliquot was used for MALDI-TOF
MS. The remaining beads were washed with stock solution of HEPBS
buffer (2 ꢀ 2 mL) and subjected to the next elongation cycle.
[14] a) J. P. Ferris, A. R. Hill Jr., R. Liu, L. E. Orgel, Nature 1996,
381, 59 – 61; b) K. J. Prabahar, T. D. Cole, J. P. Ferris, J. Am.
Chem. Soc. 1994, 116, 10914 – 10920; c) W. Huang, J. P. Ferris, J.
Am. Chem. Soc. 2006, 128, 8914 – 8919; d) P. C. Joshi, M. F.
Aldersley, J. W. Delano, J. P. Ferris, J. Am. Chem. Soc. 2009, 131,
13369 – 13374.
[15] a) G. Costanzo, R. Saladino, G. Botta, A. Giorgi, A. Scipioni, S.
Pino, E. Di Mauro, ChemBioChem 2012, 13, 999 – 1008; b) G.
Costanzo, S. Pino, F. Ciciriello, E. Di Mauro, J. Biol. Chem. 2009,
284, 33206 – 33216.
[16] O. Taran, O. Thoennessen, K. Achilles, G. von Kiedrowski, J.
Syst. Chem. 2010, 1, 9.
[17] M. Eigen, Naturwissenschaften 1971, 58, 465 – 523.
[18] J. Derr, M. L. Manapat, S. Rajamani, K. Leu, R. Xulvi-Brunet, I.
Joseph, M. A. Nowak, I. A. Chen, Nucleic Acids Res. 2012, DOI:
10.1093/nar/gks065.
Received: May 18, 2012
Published online: && &&, &&&&
[19] I. A. Chen, M. A. Nowak, Acc. Chem. Res. 2012, DOI: 10.1021/
ar2002683.
Keywords: DNA · solid-phase synthesis · primer extension ·
protecting groups · reversible terminator
.
[20] a) U. Plutowski, S. R. Vogel, M. Bauer, C. Deck, M. Pankratz, C.
Richert, Org. Lett. 2007, 9, 2187 – 2190; b) K. Giessler, H.
Griesser, D. Gçhringer, T. Sabirov, C. Richert, Eur. J. Org.
Chem. 2010, 3611 – 3620; c) C. Deck, H. Vogel, M. Jauker, C.
Richert, GIT Lab. Fachz. 2011, 778 – 780.
[1] a) A.-J. Berdis, Chem. Rev. 2009, 109, 2862 – 2879; b) E. Nudler,
Annu. Rev. Biochem. 2009, 78, 335 – 361.
[21] H. Vogel, C. Richert, ChemBioChem 2012, 13, 1474 – 1482.
[22] For two controlled extensions, see: N. Griesang, K. Giessler, T.
Lommel, C. Richert, Angew. Chem. 2006, 118, 6290 – 6294;
Angew. Chem. Int. Ed. 2006, 45, 6144 – 6148.
[23] a) S. Gryaznov, J.-K. Chen, J. Am. Chem. Soc. 1994, 116, 3143 –
3144; b) V. Tereshko, S. Gryaznov, M. Egli, J. Am. Chem. Soc.
[2] S. L. Beaucage, M. H. Caruthers, Tetrahedron Lett. 1981, 22,
1859 – 1862.
[3] a) I. A. Kozlov, L. E. Orgel, Mol. Biol. 2000, 34, 781 – 789;
b) L. E. Orgel, Crit. Rev. Biochem. Mol. Biol. 2004, 39, 99 – 123.
[4] a) M. Kurz, K. Gçbel, C. Hartel, M. W. Gçbel, Angew. Chem.
1997, 109, 873 – 876; Angew. Chem. Int. Ed. Engl. 1997, 36, 842 –
4
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!

Angewandte
Chemie
1998, 120, 269 – 283; c) most recent review: S. M. Gryaznov,
Chem. Biodiversity 2010, 7, 477 – 493.
[24] a) S. Pothukanuri, N. Winssinger, Org. Lett. 2007, 9, 2223 – 2225;
b) S. Pothukanuri, Z. Pianowski, N. Winssinger, Eur. J. Org.
Chem. 2008, 3141 – 3148.
[25] A. Kaiser, C. Richert, Synlett 2010, 2267 – 2270.
[26] a) R. Eisenhuth, C. Richert, J. Org. Chem. 2009, 74, 26 – 37;
b) C. N. Tetzlaff, I. Schwope, C. F. Bleczinski, C. A. Steinberg, C.
Richert, Tetrahedron Lett. 1998, 39, 4215 – 4218.
[27] J. Guo, N. Xu, Z. Li, S. Zhang, J. Wu, D. H. Kim, M. S. Marma, Q.
Meng, H. Cao, X. Li, S. Shi, L. Yu, S. Kalachikov, J. J. Russo, N. J.
Turro, J. Ju, Proc. Natl. Acad. Sci. USA 2008, 105, 9145 – 9150.
[28] a) D. R. Bentley et al., Nature 2008, 456, 53 – 59 (see the
Supporting Information); b) S. Balasubramanian, Angew.
Chem. 2011, 123, 12612 – 12616; Angew. Chem. Int. Ed. 2011,
50, 12406 – 12410.
[29] A. Isidro-Llobet, M. ꢁlvarez, F. Albericio, Chem. Rev. 2009, 109,
2455 – 2504.
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Communications
DNA-Directed Synthesis
A. Kaiser, S. Spies, T. Lommel,
C. Richert*
&&&& — &&&&
Template-Directed Synthesis in 3’- and 5’-
Direction with Reversible Termination
Extending both ways: A method for DNA-
templated synthesis on solid support is
described. Controlled, stepwise chain
extension was demonstrated both in the
direction favored by nature (3’-extension;
see scheme) and in the direction typical
for conventional DNA synthesizers (5’-
extension).
6
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!