3752
A. Misra / Bioorg. Med. Chem. Lett. 17 (2007) 3749–3753
GGTT) (0.25 A260 unit) dissolved in 0.1M phosphate
buffer containing 1.0 M NaCl (pH 7.5) by keeping for
HS-(CH ) OPO -d(CCAGGCAGTTCAAAATTT) and
2 6 3
0
HS-(CH ) OPO -d(CCACCGGGAATCTTTAAA)-3
2
6
3
1
h at 45 ꢁC in a hybridization chamber and then at
were immobilized on the microslide following PATH-2
in triplicate and hybridized with the complementary
room temperature overnight. After thorough washing
with phosphate buffer (3 · 15 mL) the slides were dried
and visualized under fluorescence microscope (Fig. 2).
0
fluorescent oligonucleotide probe 5 -FAM-(CH ) -OPO -d
2
3
3
(AAAACGTCGTGCTGGGTT) as mentioned above.
The fully matched duplex in microslide 1 (lane b) and
microslide 2 (lane c) has shown the fluorescence
(Fig. 3) while the unmatched complementary strands
have not generated fluorescence at all. Thus, clearly con-
firmed the specificity of the immobilization chemistry
and utility of the newly developed heterobifunctional
reagent TPMC. Encouraged with the efficiency of the
reagent and stability in the aqueous medium at pH
range between 6.5 and 7.5, further work is under
progress for the construction of microarray and
immobilization of the biomolecules like peptides,
proteins, etc. and would be communicated.
The minimum concentration of oligonucleotide required
for the immobilization on the glass surface was deter-
mined by reacting a maleimide-activated glass micro-
slide (PATH-2) with an FITC labeled oligomer d(AA
AAAAAAAAAAAAAAAA)-OPO -(CH ) SH dissolved
3
2 6
in phosphate buffer in four different concentrations (2.5,
5
, 10 and 15 lM) and after washing with buffer and
distilled water (3 · 15 mL) fluorescence intensity was
measured. It was found that the spot of 10 lM concen-
tration was sufficient for easy visualization (Fig. not gi-
ven) and hence was selected for immobilization of
oligonucleotides and its hybridization study.
To demonstrate the specificity of the proposed chemical
method for the immobilization of oligonucleotide
strands (-SH), three oligonucleotide sequences, namely,
Acknowledgment
The financial support from DST, New Delhi, is grate-
fully acknowledged.
0
5
-HS-(CH ) OPO -d(AACCCAGCACGACGTTTT);
2 6 3
References and notes
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Figure 2. Immobilization of oligonucleotide sequence 5 -HS-(CH
PO -d(AACCCA GCACGACGTTTT) via PATH-1 (slide 1) and
PATH-2 (slide 2).
2 6
) O-
3
4
5.
4
5
. Sosnowskii, R. E.; Tu, E.; Butler, W.; O’Connell, J.;
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2
1
1
1. Kumar, P.; Gupta, K. C. Bioconjug. Chem. 2003, 14, 507.
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Figure 3. Specificity of immobilization. Three oligonucleotide
0
sequences in microslide 1, viz., 5 -HS-(CH
0
2
)
6
OPO
3
-d(CCAG GCAG
TTCAAAATTT) (lane a); 5 -HS-(CH
0
2
)
OPO
6
OPO
3
-d(AACCCAG CCGA
-d(CCACCGGGA ATC
14. Broude, N. E.; Woodward, K.; Cavallo, R.; Cantor, C. R.;
Englert, D. Nucleic Acids Res. 2001, 29, e92.
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Acids Res. 2000, 28, e71.
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Nucleic Acids Res. 2000, 28, e91.
CGTTTT) (lane b) and 5 -HS-(CH
2
)
6
3
0
0
TTTAAA)-3 (lane c). And in microslide 2, viz., 5 -HS-(CH
2
)
6
OPO
OPO -d(CC
OPO -d
AAC CCAGCACGACGTTTT) (lane c). Hybridization of both
3
-d
0
(
CCAGGCAGTTCAAAATTT) (lane a); 5 -HS-(CH
2
)
6
3
0
0
ACCGGGAATCTTTAAA)-3 (lane b) and 5 -HS-(CH
2
)
6
3
(
0
microslides with 5 -FAM-(CH
GGGTT) fluorescent probe.
2
)
3
-OPO
3
-d(AAAACGTCGTGCT
17. Kumar, P.; Choithani, J.; Gupta, K. C. Nucleic Acids Res.
2004, 32, e80.