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Title

Sequence specific interstrand photocrosslinking
for effective SNP typing

Author(s)Ami, Takehiro; Ito, Katsuya; Yoshimura,
Yoshinaga; Fujimoto, Kenzo

Citation

Organic & Biomolecular Chemistry, 2007(5): 2583‑
2586

Issue Date

2007

Type

Journal Article

Text version

author

URL

http://hdl.handle.net/10119/7904

RightsCopyright (C) 2007 Royal Society of Chemistry.
Takehiro Ami, Katsuya Ito, Yoshinaga Yoshimura
and Kenzo Fujimoto, Organic & Biomolecular
Chemistry, 2007(5), 2007, 2583‑2586.
http://dx.doi.org/10.1039/b708264a ‑ Reproducedby permission of The Royal Society of Chemistry

Description

Japan Advanced Institute of Science and Technology

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Sequence Specific Interstrand Photocrosslinking for Effective SNP
TypingTakehiro Ami, Katsuya Ito, Yoshinaga Yoshimura,* and Kenzo Fujimoto*5

Receipt/Acceptance Data [DO NOT ALTER/DELETE THIS TEXT]
Publication data [DO NOT ALTER/DELETE THIS TEXT]DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT]

We describe a simple and inexpensive SNP typing method by using sequence specific interstrand
photocrosslinking via p-carbamoylvinyl phenol nucleoside. Interstrand photocrosslinking showed ahigh degree of single nucleotide specificity up to 10 3 -fold and more, and can be used in the
10 diagnostic detection of DNA sequences.

Introduction
Single nucleotide polymorphisms (SNPs) are the mostcommon form of variation in the human genome and can be
diagnostic of particular genetic predispositions toward1
15 disease.
Most methods of DNA detection involve
hybridization by an oligodeoxynucleotide (ODN) probe to its
complementary single-strand nucleic acid target leading tosignal generation.2 In as far as the detection relies on
hybridization events,3 however, such ODN probes have
20 inherent
limitations in their selectivity. Differences inhybridization efficiency vary with sequence context and areoften very small for the detection of a single base mismatch ina target strand of DNA. To attain the high sequence selectivity,
the hybridization and washing conditions need to be carefully
25 selected
to minimize any undesirable responses frommismatched hybridization probes. From this perspective,
alternative methods that do not rely on hybridization eventsare urgently required. The enzymatic ligation method 4 andchemical ligation method 5 have been developed in use for
30 engendering high specificity in sequence detection. A single
primer extension with DNA polymerase is the representative
key reaction in enzyme-coupled SNP typing.6 We have been
studying artificial DNA bases as a tool for photochemical
DNA ligation method.7 We recently reported that a modified
CV
35 ODN containing p-carbamoylvinyl phenol nucleoside (P)
could be photocrosslinked by irradiating at 366 nm withadjacent adenine residue in a [2 + 2] manner as shown in Fig.
1a.8 Photocrosslinking-coupled SNP typing is expected todemonstrate the high performance for allele discrimination.
40 Here, we describe the coupling of DNA chip technology in ainterstrand photocrosslinking via CVP as a method for SNPtyping. This photocrosslink reaction is shown to proceed withvery high sequence specificity, and can be used in the
diagnostic detection of DNA sequences.
45

Results and discussion
To demonstrate that interstrand photocrosslinking could beincorporated into platforms suitable for DNA chip
School of Materials Science, Japan Advanced Institute of Science andTechnology, Ishikawa, 923-1292, Japan. E-mail: kenzo@jaist.ac.jp; Fax:
+81 761 51 1671; Tel: +81 761 51 1671

This journal is © The Royal Society of Chemistry [year]

technologies, we constructed the DNA chip by attachingamino-labeled ODN containing CVP (S corresponds to a
50 hexa(ethylene glycol) linker fragment) as the capture strand
onto the aldehyde-modified glass surface (Fig. 1b).7 The
sequences were taken from the H-ras protooncogene (wildtype) and the activated H-ras oncogene (mutation), which has
a C → A point mutation in codon 12.9
55

Fig. 1 Structure, sequences, and progress of interstrand
photocrosslinking. (a) Structure of CV P. (b) Capture and target
sequences used in this study. (c) Strategy for the detection of single
60 nucleotide differences on a DNA chip.

We determined the feasibility of the interstrand
photocrosslinking through ODN containing CVP on a DNA
chip (Fig. 1c). A glass chip spotted with 1 µM target DNA
65 was irradiated at 366 nm for 45 min in 50 mM sodium
cacodylate buffer (pH 7.0) and 50 mM sodium chloride. Afterthe chip had been washed with deionized water at 98 °C for 5min, a phosphate-buffered saline (PBS) solution of
streptavidin-Cy3 conjugate was added to the surface, and the
70 chip was washed twice in PBS. Fluorescence signals weredetected on a microarray scanner. As shown in Fig. 2, we
measured the strong fluorescence signal of photocrosslinked
product with the completely complementary case. To
investigate the generality of sequence discrimination, we
75 constructed five target ODNs with mismatch at various
position. Results show that a single mismatch at the seventh[journal], [year], [vol], 00–00 | 1

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position in ODN context yielded very little photocrosslinked
product, with a measured fluorescence signal that is 10 3 -foldlower that the completely complementary case (Table 1).
80 Then we constructed a set of four closely related target ODNs
with a single variable base (A, T, G, or C) in the seventh
position. Most mismatches give relative fluorescence signalsof 0.1% or less, compared to the correctly matched case.

85

Fig. 2 Fluorescence intensity acquired on a microarray scanner forthe product of photocrosslinking on matched and singly mismatchedtarget ODNs.

90

Table 1 Normalized fluorescence intensity for the photocrosslinked
product of capture strand and target ODN, correctly base paired to eightODNs that differed in a single nucleotide position.
Target ODNa

Fluorescenceintensityb

ODN(C)

5´-d(ACACCGCCG)-biotin-3´

1.0 ± 0.11

ODN(4T)
ODN(5T)

5´-d(ACATCGCCG)-biotin-3´
5´-d(ACACTGCCG)-biotin-3´

0.064 ± 0.018
0c

ODN(6T)

5´-d(ACACCTCCG)-biotin-3´

0c

ODN(7T)
ODN(8T)ODN(7A)ODN(7G)

5´-d(ACACCGTCG)-biotin-3´
5´-d(ACACCGCTG)-biotin-3´
5´-d(ACACCGACG)-biotin-3´
5´-d(ACACCGGCG)-biotin-3´

0c
0c
0c
0c

Underlined character indicates the mismatched base. bEach experimenthas been repeated at least three times. c The average intensity as measured
95 is approximately equal to zero.

photocrosslinking itself and the hybridization selectivity
between the target DNA and capture strand. We also
conducted wild-type detection in the conventional method forthe same target to verify the potential selectivity of our
115 interstrand photocrosslinking method for SNP detection. A
conventional method of increasing target selectivity is to washthe DNA chip with a buffer solution at an adequate
temperature, which results in the dehybridization of DNA
duplexes formed from non-complementary strands. In the
120 wild-type detection by the traditional method, which shows
only hybridization specificity, the observed selectivity for thematched (wild-type) sequence was about 3, even after
washing with a stringent solution at the appropriate
temperature (Fig. 4a). One possible reason for the decrease in
125 the selectivity is the loss of DNA duplexes of matchedsequences, particularly during the washing step employed toremove non-complementary strands from the DNA chip. A
second possible source of low selectivity is incomplete
washing of the mismatched sequences (mutant). By contrast,
130 thefluorescence image after use of the interstrand
photocrosslinking method showed no loss of the target DNA,due to covalent bonding between the capture strand and target
DNA (Fig. 4b). Moreover, mismatched duplexes with thepotential to harm the fluorescence imaging were eliminated135 completely by high temperatures (Fig. 4c, 4d).

aFig. 3 (a) Fluorescence image for a time course of wild-type
detection. The capture strands (Y = G) were attached to the surface.
100 (b) Differing rates of photocrosslinking of matched and singly
mismatched target DNA.

As shown in Fig. 3a and 3b, results show that theinterstrand photocrosslinking reached the 50% stage in 45 min.
105 Significantly, a single mismatch in the target DNA yieldedvery little photocrosslinked product, with a measured rate thatwas 10 3 -fold lower than that of the completely complementarycase.
In such an interstrand photocrosslinking, there are two
110 possible
sourcesofspecificity:theinterstrand
2 | [journal], [year], [vol], 00–00

Fig. 4 Influence of washing and interstrand photocrosslinking on
specificity. The capture strands (Y = G) or traditional probe (5´140 d(CGGCGGTGT)-SSSS-NH 2 -3´) were attached to the surface, andtargets (left: X = C, 3 × 3 spots; right: X = A, 3 × 3 spots) wereapplied. (a) Use of only hybridization specificity and washing withbuffer under stringent conditions (25 °C). The observed specificity
was 3. (b) Use of both hybridization specificity and interstrand
145 photocrosslinking specificity and washing with buffer under stringent
conditions. The observed specificity was 13. (c) Use of only
hybridization specificity and washing with the deionized water at
98 °C for 5 min. No signal was observed. (d) Use of bothhybridization specificity and interstrand photocrosslinking specificity
150 and washing with the deionized water at 98 °C for 5 min. The
observed specificity was 52.

Finally, to demonstrate the generality of sequence
discrimination, we constructed a set of four closely related
155 targets (all in the H-ras context) with a single variable base
(A, G, C, or T), along with 9-mer immobilized capture strands
with the same four bases. We then performed kinetic
measurements for all 16 possible combinations of capture
strands and target DNAs (Table 2). As shown in Fig. 5, The
160 photocrosslinking of each of the 12 possible mismatchedsequences is presented as a ratio between theThis journal is © The Royal Society of Chemistry [year]

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photocrosslinking efficiency of each mismatched reaction andthat of the corresponding matched one. Most mismatches are
discriminated by a factor greater than 10 3 -fold, as compared to
165 the corresponding matched sequence (G-T, G-A, G-G, A-C,
A-A, C-C, T-C, T-T), and almost all of the remaining
sequences are discriminated by greater than 10 2 -fold.
Although the discrimination of G-T mismatch is moredifficult than other kinds of mismatches,10 use of the
170 interstrand photocrosslinking method exhibits the adequate
sequence specificity.
Table 2 Survey of photocrosslinking rates for all base-pairing
combinations in the context of the H-ras system.
Base pair
(Y-X)
G-C
G-T
G-A
G-G
A-C
A-T
A-A
A-G
175

Fluorescenceintensitya
1.0 ± 0.145
0b
0b
0b
0b
1.0 ± 0.042
0b0.025 ± 0.024

Base pair
(Y-X)
C-C
C-T
C-A
C-G
T-C
T-T
T-A
T-G

Fluorescenceintensitya
0b0.017 ± 0.029
0.019 ± 0.023
1.0 ± 0.0250b0b
1.0 ± 0.027
0.041 ± 0.074

aEach experiment has been repeated at least three times. bThe averageintensity as measured is approximately equal to zero.

transilluminator (FUNAKOSHI, TFL-40, 366 nm, 5,700µW/cm 2 ). HPLC was performed on a Chemcobond 5-ODS-H
column (4.6 × 150 mm) with a JASCO PU-980, HG-980-31,
DG-980-50 system equipped with a JASCO UV 970 detector
at 260 nm. The reagents for the DNA synthesizer such as A, G,
C, T-β-cyanoethyl phosphoramidite, and CPG support were
200 purchasedfrom Glen Research. Calf intestine alkaline
phosphatase (AP) was purchased from Promega. Nuclease P1
was purchased from Yamasa.
195

Preparation of ODNODN sequences were synthesized by the conventional
phosphoramidite method by using an Applied Biosystems
3400 DNA synthesizer. The coupling efficiency wasmonitored with a trityl monitor. The coupling efficiency of
crude mixture of CVP was 97% yield. The coupling time ofcrude mixture of CVP was 999 sec. They were deprotected by
210 incubation with 28% ammonia for 4 h at 65 ˚C and were
purified on a Chemcobond 5-ODS-H column (10 × 150 mm)
by reverse phase HPLC; elution was with 0.05 M ammonium
formate containing 3-20% CH 3 CN, linear gradient (30 min) ata flow rate of 3.0 mL/min. Preparation of ODNs was
11
215 confirmed by MALDI-TOF-MS analysis.
The target DNAs
were 5´-TCTGGATCAGCTGGATGGTCAGCGCACTCTTG
CCCACACCGXCGGCGCCCACCACCACCAGCTTATATTCCGTCATCGCTCCTCAGGGGCCTGCGGCCCG-biotin-3´,
where X was either A, G, C, or T.
205

220

180

Fig. 5 Fluorescence intensity for all 16 possible combinations of
capture strands and target DNAs. Each experiment has been repeatedat least three times.

Conclusions
In conclusion, we demonstrated a simple and inexpensive SNP
typing method by using sequence specific interstrand
185 photocrosslinking
via CVP. Interstrand photocrosslinking
showed a high degree of single nucleotide specificity up to
103 -fold and more. Therefore, this system can be widely used
for sensitive assays for clinical diagnostics of genetic andinfectious disease.
190

The amino-labeled ODN probe containing CVP was diluted toa concentration of 10 µM in 100 mM sodium cacodylate
buffer (pH 7.0). Spotting was accomplished by using 4 µL
aliquots from a standard micropipette. Binding of aminoCV
225 labeled ODN probe containing
P to the surface was
performed over a period of 12 h at room temperature in ahumid chamber. After probe immobilization, the glass surface
was rinsed with 0.1% SDS and deionized water. The surface
was deactivated with a solution made of NaBH 4 (3.75 mg),
230 PBS (1.5 mL), and ethanol (375 µL) over a period of 5 min.
The surface was subsequently washed with deionized water,and dried.
Interstrand photocrosslinking on a DNA chip
A glass chip spotted with a solution (4 µL) made of 1 µM
target DNA in 50 mM sodium cacodylate buffer (pH 7.0) and
50 mM sodium chloride was irradiated at 366 nm for 45 min.
After the chip had been washed with deionized water at 98 °C
for 5 min, a PBS solution of streptavidin-Cy3 conjugate (20µg/mL) was added to the surface, and the chip was washed
240 twice in PBS. Fluorescence measurements were performed ona microarray scanner CRBIO II e (Hitachi), which was
equipped with a laser with excitation wavelength of 532 nm.
235

Acknowledgements

Experimental
GeneralMass spectra were recorded on a Voyager-DE PRO-SF,
Applied Biosystems. Irradiation was performed by 25 W
This journal is © The Royal Society of Chemistry [year]

DNA probe immobilization

245

This paper is dedicated to the memory of the late Professor
Emeritus Yoshihiko Ito. This work was supported by a Grantin-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science, and Technology, Japan. This work
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was also supported by the Incubation Program of Innovation
Plaza Ishikawa from the Japan Science and Technology
250 Agency.

Notes and references
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11 MALDI-TOF-MS: calcd 4365.25 for capture strand (Y = G) [(M +H)+], found 4365.21; calcd 4340.24 for capture strand (Y = T) [(M +H)+], found 4340.18; calcd 4349.25 for capture strand (Y = A) [(M +H)+], found 4349.87; calcd 4325.22 for capture strand (Y = C) [(M +290H)+], found 4325.27; calcd 4328.19 for traditional probe [(M + H) +],found 4328.15.

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