comparable to those obtained for chemically modified RNA
0
(B90%). The labeling efficiency of the 5 -modified RNAs was
found to depend on both, the incubation time and the excess of
dansyl-tetrazine (see ESIw, Fig. S9 and S10).
To further increase the potential of the labeling approach,
we investigated the modification of RNAs of longer length. A
2
52 mer dsDNA was in vitro transcribed in the presence of
initiator nucleotide, yielding a 233nt long RNA. The DA
reaction performed with biotin–tetrazine showed efficient
labeling of this long RNA (see ESIw, Fig. S11).
In summary, we could demonstrate the successful implementa-
tion of the inverse electron-demand Diels–Alder reaction for RNA
labeling. The reaction works smoothly in neutral water without
any additives like transition metal ions, organic solvents, etc. With
only a 5-fold excess of the labeling reagent, an appreciable labeling
of RNA has been achieved. Having this RNA labeling system as a
starting point, we are currently developing more reactive dieno-
philes for a fast and quantitative labeling of RNA.
Fig. 2 LC chromatogram and mass spectra (LC-MS) of the Diels–
This work was supported by the Deutsche Forschungsge-
meinschaft (Ja 794-3), the Volkswagen foundation (VW-I/82549)
and the Fonds der Chemischen Industrie. We thank Sandra
Suhm for technical assistance and Prof. M. Wiessler (DKFZ
Heidelberg) for providing dansyl-tetrazine.
Alder reaction performed on a 19mer norbornene-modified RNA.
1
9
0 mM of RNA was incubated with a 5-fold excess of tetrazine for
0 min at RT and directly loaded to the LC-MS system.
Notes and references
1
2
J. S. Mattick, FEBS Lett., 2011, 585, 1600–1616.
P. M. Chadwick and I. Durrant, Methods Mol. Biol., 1994, 28,
101–105; M. J. Davies, A. Shah and I. J. Bruce, Chem. Soc. Rev.,
2000, 29, 97–107; J. Temsamani and S. Agrawal, Mol. Biotechnol.,
1996, 5, 223–232.
3
4
¨
B. Seelig and A. Jaschke, Chem. Biol., 1999, 6, 167–176; S. Fusz,
A. Eisenfuhr, S. G. Srivatsan, A. Heckel and M. Famulok, Chem.
Biol., 2005, 12, 941–950.
M. Hengesbach, A. Kobitski, F. Voigts-Hoffmann, C. Frauer,
G. U. Nienhaus and M. Helm, Curr. Protoc. Nucleic Acid Chem.,
2
008, 34, 11-12-1–11-12-22; A. Y. Kobitski, A. Nierth, M. Helm,
A. Jaschke and G. U. Nienhaus, Nucleic Acids Res., 2007, 35,
047–2059.
G. T. Hermanson, Bioconjugate Techniques, Academic Press,
San Diego, 1996.
6 C. R. Becer, R. Hoogenboom and U. S. Schubert, Angew. Chem.,
Int. Ed., 2009, 48, 4900–4908; C. I. Schilling, N. Jung, M. Biskup,
U. Schepers and S. Brase, Chem. Soc. Rev., 2011, 40, 4840–4871.
¨
¨
2
Fig. 3 (a) Incorporation of the initiator nucleotide by RNA poly-
merase; (b) synthetic scheme for the synthesis of a norbornene-
modified guanosine initiator nucleotide; BTT: benzylthiotetrazole,
TBAF: tetra-butylammoniumfluoride.
5
RNA and the reactivity of the RNA-appended dienophile in
Diels–Alder reactions, were confirmed by polyacrylamide
gel electrophoresis (PAGE) and ESI-MS (Fig. 4 and ESIw,
Table S2 and Fig. S8). Conversions were found to be
7 E. Paredes and S. R. Das, ChemBioChem, 2011, 12, 125–131.
8
9
M. Aigner, M. Hartl, K. Fauster, J. Steger, K. Bister and
R. Micura, ChemBioChem, 2011, 12, 47–51.
E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009, 48,
6974–6998.
1
1
0 J. C. Jewett, E. M. Sletten and C. R. Bertozzi, J. Am. Chem. Soc.,
010, 132, 3688–3690; E. M. Sletten, H. Nakamura, J. C. Jewett
and C. R. Bertozzi, J. Am. Chem. Soc., 2010, 132, 11799–11805.
1 M. L. Blackman, M. Royzen and J. M. Fox, J. Am. Chem. Soc.,
2
2
008, 130, 13518–13519; N. K. Devaraj, R. Weissleder and
S. A. Hilderbrand, Bioconjugate Chem., 2008, 19, 2297–2299;
M. Wiessler, E. Mueller, C. Kliem and H. Fleischhacker,
DE Pat., WO144200, 2007.
1
1
1
1
1
2 J. Schoch, M. Wiessler and A. Ja
32, 8846–8847.
3 C. Pitulle, R. G. Kleineidam, B. Sproat and G. Krupp, Gene, 1992,
12, 101–105.
4 J. F. Milligan, D. R. Groebe, G. W. Witherell and
O. C. Uhlenbeck, Nucleic Acids Res., 1987, 15, 8783–8798.
¨
schke, J. Am. Chem. Soc., 2010,
1
1
5 R. Fiammengo, K. Musilek and A. Ja
005, 127, 9271–9276.
6 S. Pfander, R. Fiammengo, S. I. Kirin, N. Metzler-Nolte and
A. Jaschke, Nucleic Acids Res., 2007, 35, e25; L. Zhang, L. Sun,
¨
schke, J. Am. Chem. Soc.,
Fig. 4 (a) 20% denaturing PAGE gel of the DA-reaction performed
0
2
on radioactively labeled 5 -initiated RNA; (ꢀ): initiated RNA without
dansyl-tetrazine, (+): initiated RNA (10 mM) incubated with dansyl-
tetrazine (50 mM) for 90 min in water at RT; (b) HR-ESI-MS of RNA-
Initiator and RNA-Initiator Diels–Alder product.
¨
Z. Cui, R. L. Gottlieb and B. Zhang, Bioconjugate Chem., 2001, 12,
939–948.
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12536–12537 12537