.
Angewandte
Communications
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
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Lommel, U. Plutowski, A. Hochgesand, C. Richert, Angew.
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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.
5880 – 5885; b) K. Leu, B. Obermayer, S. Rajamani, U. Gerland,
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A. Ricardo, M. Krishnamurthy, J. C. Blain, J. W. Szostak, J. Am.
Kerremans, A. Van Aerschot, R. Busson, P. Herdewijn, L. E.
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,
[15] a) G. Costanzo, R. Saladino, G. Botta, A. Giorgi, A. Scipioni, S.
[16] O. Taran, O. Thoennessen, K. Achilles, G. von Kiedrowski, J.
[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.
Griesser, D. Gçhringer, T. Sabirov, C. Richert, Eur. J. Org.
Richert, GIT Lab. Fachz. 2011, 778 – 780.
[22] For two controlled extensions, see: N. Griesang, K. Giessler, T.
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.
4
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
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