Homogeneous fluorescence assays for RNA diagnosis by pyrene-conjugated 2′-O-methyloligoribonucleotides

Article abstract of DOI:10.1080/15257770701615615

We developed a bispyrene-conjugated 2′-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701615615

Nucleosides, Nucleotides, and Nucleic Acids, 26:1659–1664, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701615615
HOMOGENEOUS FLUORESCENCE ASSAYS FOR RNA DIAGNOSIS
BY PYRENE-CONJUGATED 2ꢀ-O-METHYLOLIGORIBONUCLEOTIDES
Takashi Sakamoto, Akio Kobori, Masako Shigezawa, Yasutaka Amitani,
Maiko Higuchi, and Akira Murakami
Department of Biomolecular Engineering,
2
Kyoto Institute of Technology, Kyoto, Japan
We developed a bispyrene-conjugated 2 ꢁ-O-methyloligoribonucleotide as an RNA-specific RNA-
2
probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and dis-
criminating RNA from DNA. The assay was carried out in homogeneous aqueous media without
removing the unbound probe from the detection solution. This homogeneous fluorescence assay also
discriminated mismatch sequences in the target RNA. These pyrene probes could possess high poten-
tial to detect RNA in biological specimens simply.
Keywords Pyrene-conjugated fluorescence probe; homogeneous fluorescence assay;
RNA diagnosis; SNP
INTRODUCTION
Like proteome, transcriptome has become an important issue in the
second generation of the genome project.[1,2] The profile of mRNA expres-
sion reveals the detailed features of the genetic cascade. Thus far, analyses
have been carried out mainly by DNA chips after the amplification of target
RNAs via an RT-PCR protocol. To elucidate the transcriptome from other
viewpoints, it is desirable to directly analyze target RNAs as they are in the
living cell. We have developed fluorescent RNA probes for such analysis.[3,4]
These probes each contain one or two 2ꢁ-O-pyrenylmethyluridines in
the middle of the oligonucleotides (mono- and bispyrene-conjugated
2ꢁ-O-methyloligoribonucleotide; OMUpy and OMUpy2, respectively). Both
probes represent unique fluorescent characteristics.[3,4] The fluorescence
intensity at 375 nm of OMUpy was drastically enhanced only when it was
This research was partly supported by Grants for Regional Science and Technology Promotion
from the Ministry of Education, Science, Sports, and Culture of Japan, and by a Grant for Feasibility
Study from Innovation Plaza Kyoto. The authors also are indebted to Otsuka Pharmaceutical Co., Ltd.,
for their financial support.
Address correspondence to A. Murakami, Department of Biomolecular Engineering, Kyoto Insti-
tute of Technology, Matsugasaki, Kyoto 606-8585, Japan. E-mail: akiram@kit.ac.jp
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hybridized with its complementary oligoribonucleotides (cORN), and
was scarcely enhanced when hybridized with its complementary DNA
(cODN).[4] OMUpy2 showed broad fluorescence around 480 nm when
hybridized with cORN, and barely showed fluorescence when hybridized
with cODN. These results suggest that OMUpy2 could be used to visu-
ally detect specific RNAs in homogeneous media.[5] However, there is
a limitation in the design of the probe sequences. That is, each target
site should contain consecutive AA sites. In this proceeding, we report
the development and characteristics of novel bispyrene-conjugated 2ꢁ-
O-methyloligoribonucleotides that contain 2ꢁ-O-pyrenylmethylcytidine
and/or 2ꢁ-O-pyrenylmethyluridine, denoted as OMCpy2, OMUpyCpy, and
OMCpyUpy, respectively.
MATERIALS AND METHODS
Preparation of 2ꢁ-O-pyrenylmethylcytidine (Cpy)[6]
2ꢁ/3ꢁ-O-(pyrenylmethyl)cytidine (2). Sodium hydride (60%) (600 mg,
15 mmol) was added to a solution of cytidine (3.65 g, 15 mmol) in dry
dimethylformamide (150 mL) at 5◦C under N2 atmosphere. After the mix-
ture was stirred for 1 hour, chloromethylpyrene (1.25 g, 5 mmol) was added
and the reaction was maintained at 70◦C for 16 hours. Ice was then added
and the reaction mixture was concentrated near dryness. After 50 mL of
CHCl3 and 50 mL of water were added to the residue, the insoluble solid
was filtered, washed twice with cooled water and CHCl3, and dried in vacuo.
A mixture of 2ꢁ/3ꢁ-isomers (495 mg, 22%) was obtained as brown powder.
4-N-benzoyl-2’/3’-O-(pyrenylmethyl)cytidine(3)
A mixture of 2ꢁ/3ꢁ-isomers of 2ꢁ/3ꢁO-pyrenylmethylcytidine (2) (228 mg,
0.5 mmol) was dried by repeated coevaporation with pyridine and sus-
pended in 2.5 mL of dry pyridine. To the solution was added 317 µL
(2.5 mmol) of trimethylchlorosilane. After the mixture was stirred for
15 minutes, 290 µL of benzoyl chloride (2.5 mmol) was added and the
reaction was maintained at room temperature for 2 hours. The mixture was
then cooled in an ice bath and 0.5 mL of water was added. After 5 minutes,
0.5 mL of 29% aqueous ammonia was added and the mixture was stirred at
room temperature for 0.5 hour. The reaction was then evaporated to near
dryness and the residue was dissolved in 50 mL of CHCl3-MeOH (3:1, v/v).
The solution was washed three times with 30 mL of water. The organic layer
was dried over Na2SO4, filtered, and concentrated to dryness. The residue
was purified by flash chromatography to give 2ꢁ/3ꢁ-isomers of 3(127 mg,
45%) as brown powder.

Homogeneous Fluorescence Assays
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4-N-benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-O-(pyrenylmethyl)
cytidine (4)
A mixture of 2ꢁ/3ꢁ-isomers of 4-N -benzoyl-2ꢁ/3ꢁ-O-(pyrenylmethyl) cyti-
dine(3) (400 mg, 0.71 mmol) was dried by repeated coevaporation with
pyridine and suspended in 10 mL of dry pyridine. To the solution was
added 4,4’-dimethoxytritylchloride (290 mg, 0.86 mmol), and the mixture
was stirred at room temperature for 12 hours. The mixture was concen-
trated to half volume, diluted with CHCl3, and washed three times with 5%
NaHCO3(aq). The organic layer was dried over Na2SO4, filtered, and con-
centrated to dryness. The residue was purified by flash chromatography to
give 4 (515 mg, 84%) as white foam.
Preparation of Bispyrene-Conjugated
2’-O-methyloligoribonucleotides
Bispyrene-conjugated
2ꢁ-O-methyloligoribonucleotides
(OMCpy2,
OMUpyCpy, OMCpyUpy) were synthesized according to the reported
procedure.[3] Fluorescence spectra were obtained by a spectrophotome-
ter (RF-5300PC, Shimadzu, Japan) equipped with a thermal controller.
Equimolar solutions of OMUpy2 (0.75 µM) and corresponding ORN (0.75
µM) were denatured at 75◦C for 5 minutes and slowly cooled to 11◦C prior
to measurements in 10 mM sodium phosphate (pH 7.0), 0.1 M NaCl.
RESULTS AND DISCUSSION
The synthesis of the Cpy phosphoramidite unit is shown in
Scheme 1. The synthetic route begins with 2ꢁ-O-alkylation of cytidine us-
ing chloromethylpyrene. A mixture of the 2ꢁ,3ꢁ-isomers was obtained, and
the pure 2ꢁ-product was not isolated until the DMT stage. The ratio of
the 2ꢁ-isomer to the 3ꢁ-isomer, estimated from the intensity of the 1’-H
1
proton of 2ꢁ/3ꢁ-O-(pyrenylmethyl)cytidine using H NMR, was 5:1. Sub-
sequent protection of the amino group of cytosine with the benzoyl
group, followed by the addition of a dimethoxytrityl group at the 5ꢁ-O-
position, gave compound 4. Conversion to phosphoramidite using chloro(2-
cyanoethoxy)(N ,N -diisopropylamino)phosphine provided a Cpy phospho-
ramidite unit.
OMCpy2, OMUpyCpy, and OMCpyUpy, denoted as RNA probes (Figure
1), each contained one or two 2ꢁ-O-pyrenylmethylcytidines inside the ORN
sequence. The fluorescence intensity of each probe around 375 nm was
less than 5% that of 1-pyrenemethanol, showing that the fluorescence due
to the pyrenes was greatly quenched. In contrast, the hybrid of the probe
with cORN showed eminent and structureless fluorescence around 480
nm, as shown in Figure 2, as well as slight fluorescence appearing around

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SCHEME 1 Reagents and conditions : a) (1) NaH (3 equiv.) DMF, 5◦C, 1 hour; (2) chloromethylpyrene
(1 equiv.), 70◦C, 16 hours; b) (1) TMSCL (5 equiv.), pyridine, (2) BzCl, (5 equiv.), pyridine, (3) NH3,
c DMTrCl (1.2 equiv.), pyridine, d chloro(2-cyanoethoxy)-(N,N-diisopropylamino)phosphine (2 equiv.)
diisopropylethylamine (5 equiv.), CHCl2, 1.5 hours.
FIGURE 1 Bispyrene-conjugated 2ꢁ-O-methyloligoribonucleotide.

Homogeneous Fluorescence Assays
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FIGURE 2 Fluorescence spectra of OMCpy2, OMUpyCpy, and OMCpyUpy in the presence of the com-
plementary oligoribonucleotide. (a) OMCpy2, (b) OMUpyCpy, (c) OMCpyUpy Dashed line: OMCpy2;
OMUpyCpy; OMCpyUpy, Solid line: OMCpy2 + cORN; OMUpyCpy + cORN; OMCpyUpy + cORN.
[OMCpy2] = [OMUpyCpy] = [OMCpyUpy] = [ORN] = 0.75 µM Temperature: 11◦C.
375 nm, suggesting that the pyrenes relocated from the regions between
nucleobases to the outside of the duplex and consequently interacted with
each other. Interestingly, the fluorescence intensity of the hybrid of the
probe with cODN was scarcely changed, suggesting that the pyrenes in the
duplex of OMUpy2 and cODN remained intercalated between nucleobases.
These results were substantially similar to the characteristics of OMUpy2,
while the fluorescence intensities varied depending on the kind of pyrene-
conjugated nucleoside. The difference could be attributed to the local du-
plex structure near the pyrene moieties. It should be noted that these RNA
probes are RNA-specific fluorescence probes and that they are applicable to
gene diagnosis.
FIGURE 3 Fluorescence intensity at 480 nm of OMCpy2 in the presence of the oligoribonucleotides.
ORNs sequence: r ( GGUGGUGAAGCXGGCG); ORNs sequence: r ( GGUGGUGAAGCAXGCG).

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T. Sakamoto et al.
The characteristics of the probes were then applied to discriminate mis-
match sequences in ORNs. Figure 3 shows the results of the case of OMCpy2.
Fluorescence was scarcely observed when the mismatch site was located in
the vicinity of the Cpy residue, and increased as the mismatch site grew apart
from the Cpy residue. These results suggest that the pyrene excimer (or
pyrene dimer) formed only when the pyrene-conjugated nucleoside was in-
corporated in the firm duplex, and that these bispyrene-conjugated oligonu-
cleotides could be unique and prominent RNA probes to detect SNPs. The
probe was then immobilized on solid matrix such as a glass plate to fabricate
an RNA chip, which was able to detect the complementary ORN without a
washing protocol. Further study is underway.
CONCLUSION
We have developed a novel fluorescent oligonucleotide probe that can
detect specific RNA and can discriminate a singly altered sequence of RNA.
Future development of the probe should enable its use in living cells. For
this purpose, we have chosen phosphorothioate linkages to construct the
probes, and this work is under way.
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2ꢁ-O-methyloligonucleotide as a highly specific RNA-recognition probe. Angew. Chem. Int. Ed. 2002,
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