A new concept for DNA-arrays

Article abstract of DOI:10.1081/NCN-120023015

We will insert a cleavage site in an oligodeoxynucleotide, which can be used for a selective and quantitative cleavage. For that reason we synthesized the four 5′-S-(4,4′-dimethoxytrityl)-mercapto-2′ -deoxynucleotide-3′-O-(2-cyanoethoxydiisopropylamino)-p

Full text of DOI:10.1081/NCN-120023015

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A New Concept for DNA-Arrays
Kerstin Jahn-Hofmann a , Nancy Holzhey b , Thomas Ellinger b & Joachim W. Engels a c
a Institut für Organische Chemie und Chemische Biologie (OCCB), Johann-Wolfgang-Goethe
Universität , Frankfurt am Main, Germany
b Firma Clondiag Chip Technologies GmbH , Jena, Germany
c Institut für Organische Chemie, Johann-Wolfgang-Goethe Universität , Mertonviertel AK
Engels N160, Marie-Curie-Strasse 11, D-60439, Frankfurt, Germany
Published online: 31 Aug 2006.
To cite this article: Kerstin Jahn-Hofmann , Nancy Holzhey , Thomas Ellinger & Joachim W. Engels (2003) A New Concept for
DNA-Arrays, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1479-1482, DOI: 10.1081/NCN-120023015
To link to this article: http://dx.doi.org/10.1081/NCN-120023015
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1479–1482, 2003
A New Concept for DNA-Arrays
Kerstin Jahn-Hofmann,1 Nancy Holzhey,2 Thomas Ellinger,2
and Joachim W. Engels1,
*
1Institut fur Organische Chemie und Chemische Biologie (OCCB),
¨
Johann-Wolfgang-Goethe Universita¨t,
Frankfurt am Main, Germany
2Firma Clondiag Chip Technologies GmbH, Jena, Germany
ABSTRACT
We will insert a cleavage site in an oligodeoxynucleotide, which can be used for a
selective and quantitative cleavage. For that reason we synthesized the four 50-S-
(4,40-dimethoxytrityl)-mercapto-20-deoxynucleotide-30-O-(2-cyanoethoxydiisopro-
pylamino)-phosphites (5a–d). The cleavage of P-S and C-S bonds is described
(Mag, M.; Lucking, S.; Engels, J.W. Synthesis and selective cleavage of an oligo-
¨
deoxy-nucleotide containing a bridged internucleotide 50-phosphorthioate link-
age. Nucleic Acids Res. 1991, 19 (7), 1437–1441; Marriott, J.H.; Mottahedeh,
M.; Reese, C.B. 9-(4-methoxyphenyl)xanthen-9-thiol: A useful reagent for the
preperation of thiols. Tetrahedron Lett. 1990, 31 (51), 7485–7488; Divakar,
K.J.; Mottoh, A.; Reese, C.B.; Shanghvi, Y.S. Approaches to the synthesis of
20 thio analogues of pyrimidine ribosides. J. Chem. Sc., Perkin Trans. 1 1990,
969–974). The oligodeoxynucleotides with an achiral bridged 50-phosphorothioate
linkage 50-O-P-S-30 are synthesized by the phosphoramidite procedure.
*Correspondence: Joachim W. Engels, Institut fur Organische Chemie, Johann-Wolfgang-
¨
GoetheUniversita¨t,Mertonviertel AK Engels N160, Marie-Curie-Strasse 11, D-60439 Frank-
furt, Germany; Fax: +49 697 982 9148; E-mail: joachim.engels@chemie.uni-frankfurt.de.
1479
DOI: 10.1081/NCN-120023015
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1480
Jahn-Hofmann et al.
Figure 1. Concept of our new DNA-array.
With the completion of the human genome project, we are just at the beginning
of a new time in genetic analysis. Wide scale DNA testing requires the development
of fast, accurate, small, cheap and easy-to-use devices. We will create a new array
system, which works with speed, is simple, has a high performance and low cost.
The actual commercial standard tests are based on three different steps: the target-
labeling, the hybridization with an immobilized probe, and the detection of the
hybridization (target-probe).[1]
We are developing an array, which works without any target-labeling. Thus we
modified the immobilized probe with a marker (label) for detection and moreover we
have a control about the quality of the chip. Furthermore we inserted a cleavage site,
which can be used for a selective and quantitative cleavage. During this cleavage the
target should protect the section around the cleavage site, Fig. 1. shows this concept.
The cleavage of P-S and C-S bonds is described and we prefer the thio-modification
because of it’s electronic and steric similarity with the natural congener and the
Figure 2. Shows the synthesis of 50-S-(4,40-dimethoxytrityl)-mercapto-20-deoxynucleotides-
30-O-(2-cyanoethoxy-diisopropylamino)-phosphites (5a-d) (x ¼ leaving group). (a) Dichloro-
acetic acid, DCM, H2S, 3 h, 4ꢀC, 95%. (b) 50-X-20-deoxynucleotide (3a-g), DMSO, 1,1,3,3-
tetramethyl-guanidine, argon, rt, (yield shows table 1.). (c) 2-cyanoethyl-N,N0-diisopropyl-
chloro-phosphoramidite, DIPEA, DCM:ACN, 1 h, rt, (yield shows Table 1.)

New Concept for DNA-Arrays
Table 1. Yields of some reactions from Fig. 1.
1481
X ¼ Leaving
B ¼ Nucleoside
Group Yield 3 a–g
Yield 4 a–g
Yield 5 a–d
(5a) 73%
T
T
T
Abz
Abz
Cbz
Gibu
(3a) Chloro 72.6%
(3b) Tosyl 66%
(3c) Mesyl 64%
(3d) Tosyl 39%
(3e) Mesyl 79%
(3f) Mesyl 48%
(3g) Mesyl 93%
(4a) 63.6%
(4b) 94%
(4c) 97%
(4d) 94%
(4e) 41%
(4f) 82%
(4g) 45%
(5b) 72%
(5c) 72%
(5d) 76.4%
opportunity of a later derivatisation. We synthesized the four 50-S-(4,40-dimethoxy-
trityl)-mercapto-20-deoxynucleotides-30-O-(2-cyanoethoxy-diisopropylamino)-phos-
phites (5a-d), see Fig. 2. and Table 1.[2] To synthesize an oligodeoxynucleotide with
an achiral bridged 50-phosphorothioate linkage 50-O-P-S-30 we used the solid phase
phosphoramidite procedure.
We started our work with the following model oligodeoxynucleotide: 50-AGC
CCT TAC TTT GAC GGT ATA TCT-30(T ¼ 50-S-(4,40-DMTr)-mercapto-20-deoxy-
thymidine-30-O-(2-cyanoethoxy-diisopropylamino)-phosphite).
The model oligodeoxynucleotide was synthesized by the phosphoramidite
method on CPG material or directly on the surface of a chip. The coupling time
for the modified T-Amidite amounts 2 ꢁ 300 sec. (CPG) or 900 sec. (chip). After
deblocking (T-Amidite) a reduction with 50 mM DTT solution is carried out to
avoid the disulfide bond formation. The first tests of the cleavage were made in solu-
tion. For these tests we incubated the CPG material with ammonia (24 h, rt) and a
preparative HPLC followed. Then we carried out several tests for the cleavage
detected by HPLC and gel electrophoresis. 50 mM AgNO3 solution cleaves the oligo-
deoxynucleotide in solution and on a surface of a biacore chip within 5 min comple-
tely. Then we performed the synthesis and the cleavage on the chip. During the
treatment with silver nitrate all positions on the chip with a modified building
block will be cleaved. The result is a free 50-thiol and we postulate, that the target
Figure 3. Shows the fluorescence images of arrays after hybridization of Cy3-labeled match
oligodeoxynucleotides (P ¼ Phosphate, PT ¼ Phosphothioate modified T-amidite) a) reference
probe (no cleavage), b) hybridization after cleavage by silver nitrate.

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Jahn-Hofmann et al.
Figure 4. Shows the fluorescence intensity before and after treatment with silver nitrate.
oligonucleotide can hold the oligonucleotide with the fluorescence label, for the
subsequent detection. There is not a distinction between a double-strand or single-
strand domain during the cleavage.
Chip Technology
Array synthesis. Carried out with ClondiagÕ micro wet printing technology
(16 stripes)
a. 4 stripes of fully matched sequence with phosphate backbone (P) 50-AGC
CCT TAC TTT GAC GGT ATA TCT-30.
b. 4 stripes of fully matched sequence with modified phosphothioate backbone
(PT) 50-AGC CCT TAC TTT GAC GGT ATA TCT-30.
c. 8 stripes of one base deletion sequence with phosphate backbone (on array
between P and PT as control).
Selective cleavage of 50-phosphorothioate linkage: ) 40 mM aqueous silver
nitrate solution, 30 minutes at room temperature (Fig. 4).
Hybridization conditions: ) 10 nM 50-AGA TAT ACC GTC AAA GTA AGG
GCT-30(50-Cy3 labeled) in 6 ꢁ SSPE, 0.1% SDS buffer, 1 h at 50ꢀC (Fig. 3).
REFERENCES
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State of the nature. Nature Biotechnology 1998, 16, 40–44.
2. Mag, M.; Lucking, S.; Engels, J.W. Synthesis and selective cleavage of an oligo-
¨
deoxy-nucleotide containing a bridged internucleotide 50-phosphorthioate linkage.
Nucleic Acids Res. 1991, 19 (7), 1437–1441; Marriott, J.H.; Mottahedeh, M.;
Reese, C.B. 9-(4-methoxyphenyl)xanthen-9-thiol: A useful reagent for the prepera-
tion of thiols. Tetrahedron Lett. 1990, 31 (51), 7485–7488; Divakar, K.J.; Mottoh,
A.; Reese, C.B.; Shanghvi, Y.S. Approaches to the synthesis of 20 thio analogues of
pyrimidine ribosides. J. Chem. Sc., Perkin Trans. 1 1990, 969–974.