Chemistry Letters Vol.33, No.12 (2004)
1555
This general characteristic of 9 indicates that any polypeptides
and proteins would be able to be introduced into the desired
and specified region of a DNA via the branched-type NAPs.
When the connection of a peptide strand and a nucleotide strand
is required, all one has to do is just to couple compound 9 or its
phosphoramidite derivative with free polypeptides or free pro-
teins. However, when the coupling at the amino terminus of a
peptide strand is required, the side residues of the polypeptide
may have to be protected.12 Especially, for the successive syn-
thesis of a DNA strand, the protecting group of the polypeptide
must not be removed by acidic treatment during the 50 deprotec-
tion of the oligonucleotide on the solid support. In this study, we
have also synthesized an NAP (His6-NAP, 12) coupled with the
octapeptide, glycine-glycine-histidine-histidine-histidine-histi-
dine-histidine-histidine (Gly2His6). Compound 12 was convert-
ed to the corresponding phosphoramidite 15 and introduced into
a DNA strand. To show one of the applications of the functional
linkage of the polypeptide and oligonucleotide, a modified oligo-
nucleotide, namely His6-NAP-T20 with a His6-NAP at the 50 end
of the eicosathymidylic acid was prepared. This His6-NAP-T20
compound is the result of the linkage of two functions: one is
the oligopeptide function to bind to the imino-diacetic acids
via divalent metal ions13 and the other is the oligonucleotide
function to bind to the polyadenylic acids (poly(A)) by Watson–
Crick base pairing. By using this molecule, one of the most use-
ful columns in molecular biology, that is the column to pick up
messenger RNA with the poly(A) tail from the total cell extract,
should be easily prepared.14
of the His6-NAP-T20 function at the oligonucleotide part. Once
the histidines were dissociated from the column resin by chela-
tion of the Ni2þ ions, the trapped complex of His6-NAP-T20 and
poly(A) was eluted from the column as shown Figure 1a. Thus,
we have succeeded in introducing an oligopeptide function into
the DNA via linkage of the oligopeptide strand and the DNA
strand by an NAP. Though the coupling of the peptide function
might be achieved by other methods previously reported,4–6 our
NAP strategy should be most common and easiest way to intro-
duce varied functions of desired amino acids or oligopeptides
into a DNA.
This work was supported in part by Grant-in-Aid from the
Ministry of Education, Culture, Sports, Science and Technology,
Japan.
References and Notes
1
a) T. Ohmichi and N. Sugimoto, Biochemistry, 36, 3514
(1997). b) T. R. Cech, Science, 236, 1532 (1987).
2
J. M. Burke, M. Belfort, T. R. Cech, R. W. Davis, R. J.
Schweyen, D. A. Shub, J. W. Szostak, and H. F. Tabak, Nucleic
Acids Res., 15, 7217 (1987).
3
4
R. Tan and A. D. Frankel, Proc. Natl. Acad. Sci. U.S.A., 92,
5282 (1995).
a) T. R. Battersby, D. N. Ang, P. Burgstaller, S. C. Jurczyk,
M. T. Bowser, D. D. Buchanan, R. T. Kennedy, and
S. A. Benner, J. Am. Chem. Soc., 121, 9781 (1999). b) S. W.
Santoro, G. F. Joyce, K. Sakthivel, S. Gramatikova, and
C. F. Barbas, III, J. Am. Chem. Soc., 122, 2433 (2000). c) D.
Forget, D. Boturyn, E. Defrancq, J. Lhomme, and P. Dumy,
Chem.—Eur. J., 7, 3976 (2001). d) D. M. Perrin, T. Garestier,
The results of the poly(A) trapping experiment by the His6-
NAP-T20 are shown in Figure 1.15 As shown in Figure 1a, the
His6-NAP-T20 was bound to a column in which the imino-diac-
etic acids were immobilized while T20 without Gly2His6 did not
bind and all the T20 passed through the column (Figure 1b). This
phenomenon implies the exhibition of the His6-NAP-T20 func-
tion at the peptide part. When the poly(A) was loaded to the col-
umn, the poly(A) molecules were then trapped on the column in
which the His6-NAP-T20 was immobilized while no trapping of
poly(A) was observed in the column with immobilized Gly2His6
without the T20 (Figure 1c). This result indicates the exhibition
´ `
and C. Helene, J. Am. Chem. Soc., 123, 1556 (2001).
a) J. C. Morales and E. T. Kool, J. Am. Chem. Soc., 122, 1001
(2000). b) O. Thum, S. Jager, and M. Famulok, Angew. Chem.,
¨
Int. Ed., 40, 3990 (2001).
a) U. Diederichsen and C. M. Biro, Bioorg. Med. Chem. Lett.,
10, 1417 (2000). b) N. Minakawa, Y. Ono, and A. Matsuda,
J. Am. Chem. Soc., 125, 11545 (2003).
5
6
7
8
9
P. E. Nielsen, M. Egholm, R. H. Berg, and O. Buchardt,
Science, 254, 1497 (1991).
T. H. Smith, M. A. Kent, S. Muthini, S. J. Boone, and P. S.
Nelson, Nucleosides Nucleotides, 15, 1581 (1996).
Synthetic protocols are available in supporting information
pdf
10 The molecular model of the sugar moiety in compound 10 is
shown in S.info as Figure S1. The torsion angles of product
10 are also listed in Figure S1 along with the values of 8.
11 Procedures for purification and quantification of the oligonu-
cleotides are stated in S.info. Themodynamic data for a duplex
containing a Leu-NAP are also available in S.info.
12 T. Wada, N. Minamimoto, Y. Inaki, and Y. Inoue, J. Am.
Chem. Soc., 122, 6900 (2000).
13 a) C. Dietrich, O. Boscheinen, K. D. Scharf, L. Schmitt, and R.
Tampe, Biochemistry, 35, 1100 (1996). b) R. Colangeli, A.
Heijbel, A. M. Williams, C. Manca, J. Chan, K. Lyashchenko,
and M. L. Gennaro, J. Chromatogr., B, 714, 223 (1998).
14 H. Gu, J. D. Gupta, and D. R. Schoenberg, Proc. Natl. Acad.
Sci. U.S.A., 96, 8943 (1999).
Figure 1. Poly(A) trapping experiment.15 Three samples, (a)
NAP-DNA (His6-NAP-T20), (b) DNA (T20), and (c) peptide
(Gly2His6) were used for the analysis. Arrows indicate the time
for loading of the solutions to the column. The UV absorption at
254 nm indicates the elution of nucleic acids (T20 or poly(A))
from the column.
15 Detailed procedure and a schematic illustration of the poly(A)
trapping experiments are shown in S.info as Figure S2.
Published on the web (Advance View) November 6, 2004; DOI 10.1246/cl.2004.1554