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Title

Highly sequence specific RNA terminal labeling by
DNA photoligation

Author(s)Yoshimura, Yoshinaga; Noguchi, Yuuki; Fujimoto,
Kenzo

Citation

Organic & Biomolecular Chemistry, 5(1): 139‑142

Issue Date

2007

Type

Journal Article

Text version

author

URL

http://hdl.handle.net/10119/7901

RightsCopyright (C) 2007 Royal Society of Chemistry.
Yoshinaga Yoshimura, Yuuki Noguchi and Kenzo
Fujimoto, Organic & Biomolecular Chemistry, 5(1),2007, 139‑142. http://dx.doi.org/10.1039/b615715g‑ Reproduced by permission of The Royal Societyof Chemistry

Description

Japan Advanced Institute of Science and Technology

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www.rsc.org/[journal] | [journal name]Highly sequence specific RNA terminal labeling by DNA photoligation†
Yoshinaga Yoshimura,a,b Yuuki Noguchib and Kenzo Fujimoto*a,b
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Publication data [DO NOT ALTER/DELETE THIS TEXT]
5 DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT]
We report the nonenzymatic terminal labeling of oligoribonucleotide (ORN) by using templatedirected photoligation through 5-carboxyvinyl-2´-deoxyuridine (CVU) with high selectivity.

Introduction
Terminal labeling of RNA by using ligation methods is an
important technique in both basic and applied sciences.
Template-directed ligation has potential applications in RNAbased biotechnologies,1-3 particularly as tools for preparing
the chimeric RNA–DNA strands, and for studying RNA
structural and functional analyses. For example, detection of
15 bacterial
RNAs with single nucleotide resolution was
performed by template-directed ligation.4 Thus, terminallabeling of RNA has become a useful method for monitoringand investigating RNA. Although DNA ligases are useful forthe ligation of DNA, enzymatic ligation methods for terminal
20 labeling of RNA face limitations. For example, DNA ligases
show low activity with RNAs.5,6 In addition, ligase methods
are not likely to be useful in intact cellular or tissue
preparations, because it would be difficult to deliver the ligase
into cells. Synthesis of chimeric RNA–DNA strands such as
25 lariat
RNAs have been reported; nevertheless, themethodologies require the use of cyanogen bromide which is ahighly toxic and irritating reagent.7 In our previously work,
we reported on the template-directed reversible photoligation
with 5-vinyl-2´-deoxyuridine (VU).8 The advantage of using
30 photons as reagents for initiating photoligation is that no
additional chemical reagents need to be introduced into thesystem. A recent photoligation method by using the RNA
template showed higher activity than the corresponding DNA
template.9 Here, we report on a terminal labeling of ORN by
CV
35 using template-directed photoligation throughU. We also
describe the high selectivity of terminal labeling of ORN by
using solid-state assays.
10

ODN(CVU) and ORN(U) (5´-r(UGUGCU)-3´) were irradiatedat 366 nm for 32 min in the presence of template ODN(A) (5´d(CACGCAAGCACA)-3´; Fig. 1b), HPLC showed theappearance of a peak relating to ORN(U)^ODN(CVU) in 96%
CV
50 yield along with the disappearance of the ODN(U) andORN(U) peaks (Fig. 2). MALDI–TOF–MS indicates that theisolated
ORN(U)^ODN(CVU)
obtainedfromHPLC
purification was a photoligated product of ODN(CVU) andORN(U) (calcd 3731.43 for [M + H]+; found 3731.15).
CV
55 Enzymatic digestion of isolated ORN(U)^ODN(U) showed
the formation of rC, rU + dC, rG, dG, and dT in a ratio of
1:3:2:3:1 together with rU– CVU photoadduct, which was
confirmed by MALDI–TOF–MS (calcd 542.1734 for [M +H]+; found 542.1682). As shown in Fig. 3, the CD spectrum ofCV
60 ORN(U)^ODN(U) and ODN(A) showed a spectrum
intermediate between B-form duplex and A-form duplex.10 On
the other hand, when ODN(CVU) and ORN(C) (5´r(UGUGCC)-3´) were irradiated at 366 nm for 32 min in thepresence of template ODN(G) (5´-d(CACGCAGGCACA)-3´),CV
65 we observed a peak corresponding to ORN(C)^ODN(U), asdetermined by HPLC.11 As shown in Fig. 4, ORN containing apyrimidine base at the 3´ terminal site reacted withphotoexcited CVU to produce a terminal modified product, but
the photoligation rates by using ORN(C) were slower than the
70 corresponding
ORN(U).
Therefore,
template-directed
photoligation methods were successful at modifying the 3´terminal site of RNA.

Results and discussion
An oligodeoxynucleotide (ODN) containing CVU, ODN(CVU)CV
40 (5´-d(
UGCGTG)-3´), was synthesized by using the
cyanoethylphosphoramidite of CVU according to conventional
DNA synthesis (Fig. 1a).8 ODN(CVU) was characterized by
nucleoside composition and MALDI–TOF–MS (calcd 1878.27
for [M + H]+; found 1878.13). We determined the feasibilityCV
45 of terminal labeling of RNA through ODN(U). WhenaSchool of Materials Science, Japan Advanced Institute of Science andTechnology, Ishikawa 923-1292, Japan. Fax: +81 761 51 1671; Tel: +81
761 51 1671; E-mail: kenzo@jaist.ac.jpb
PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012,
Japan.
† Electronic Supplementary Information (ESI) available: Experimental
details. See http://dx.doi.org/10.1039/b000000x/This journal is © The Royal Society of Chemistry [year]

75

Fig. 1 (a) Structure of 5-carboxyvinyl-2´-deoxyuridine, CVU. (b)Schematic illustration of DNA photoligation at RNA terminal.

We investigated the thermal stability of the RNA–
DNA/DNA
duplexes
containing
terminal
modifiedCV
80 ORN(U)^ODN(U) by monitoring the melting temperature(T m). In T m measurements of the duplex, sigmoidal curves on
the change of A 260 were obtained, and the T m value wascalculated from the first part of the curve (Table 1). The T mvalue (21.7 °C) of terminal modified ORN(U)^ODN(CVU) and
85 ODN(A) was lower than that of the dodecamer duplex[journal], [year], [vol], 00–00 | 1

CREATED USING THE RSC ARTICLE TEMPLATE (VER. 2.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILSORN(U)pODN(T)(5´-r(UGUGCU)d(TGCGTG)-3´)andODN(A) (50.9 °C), whereas the duplex was 7.0 °C more
thermally stable than a mixture of ORN(U), ODN(CVU), andthe template ODN(A) (14.7 °C).
90

Fig. 2 HPLC analysis of the irradiated ODN( CVU) and ORN(U) in thepresence of template ODN(A): (a) before irradiation; (b) after
irradiation at 366 nm for 32 min, 96% yield.
95

ODN containing CVU, amino-ODN(CVU) (5´-d(CVUGCGTG)SSSS-NH2 -3´; here S corresponds to a hexa(ethylene glycol)
linker fragment), onto the aldehyde-modified glass surface.8,9
115 A glass chip spotted with 2 µM template ODN(A) and biotinlabeled B-ORN(U) (5´-biotin-r(UGUGCU)-3´), was irradiatedat 366 nm for 1 h in 50 mM sodium cacodylate buffer (pH7.0) and 100 mM sodium chloride (Fig. 5). After the chip had
been washed with deionized water at 98 °C for 5 min, a
120 phosphate-buffered saline (PBS) solution of streptavidin-Cy3
conjugate was added to the surface, and the chip was washedtwice in PBS. Fluorescence signals were detected on a
microarray scanner. When amino-ODN(CVU) and B-ORN(U)were irradiated at 366 nm in the absence of template ODN(A),125 no terminal modified product was observed (Fig. 6, Lane 2).
On the other hand, we measured the strong fluorescence signalof the terminal modified product with the completely
complementary case (Fig. 6, Lane 3). Then we constructed a
set of four closely related ORNs with a single variable base
130 (A, U, G, or C) in the third position. The results show that asingle nucleotide difference at the third ORN position yieldedvery little terminal modified product, with a measured
fluorescence signal that was 32-fold lower than thecompletely complementary case (Table 2). Arylazide
135 mediated photocrosslinking of RNA has been reported, but thephotogenerated intermediate have broad, indiscriminantFig. 3 CD spectra of ORN(U)^ODN( CVU), and template ODN(A)(solid line), and ORN(C)^ODN( CVU), and template ODN(G) (brokenline).
100

140

Fig. 5 Schematic illustration for investigating the selectivity ofterminal labeling of ORN.

Fig. 4 Time course of photochemical terminal ligation of ORN(U) or
ORN(C).

105

Table 1 Tm values (°C) for duplexes.a
Tm (°C)ODN(CVU), ORN(U)/ODN(A)ORN(U)^ODN(CVU)/ODN(A)ORN(U)pODN(T)/ODN(A)ODN(CVU), ORN(C)/ODN(G)ORN(C)^ODN( CVU)/ODN(G)ORN(C)pODN(T)/ODN(G)

14.7
21.7
50.9
22.0
40.6
58.5

a

All Tm values of the duplexes (2.5 µM) were measured in 50 mM sodium
cacodylate buffer (pH 7.0) and 100 mM sodium chloride.
ORN(C)pODN(T) = 5´-r(UGUGCC)d(TGTGCT)-3´.

110

To investigate the selectivity of terminal labeling of ORN,
we constructed the DNA chip by attaching amino-labeled
2 | [journal], [year], [vol], 00–00

Fig. 6 Fluorescence intensity (top) and images (bottom) acquired on a
microarray scanner for the product of photoligation. Lane 1, ODN(A),145 irradiation at 366 nm; Lane 2, B-ORN(U), irradiation at 366 nm;
Lane 3, B-ORN(U) + ODN(A), irradiation at 366 nm; Lane 4, BORN(A) + ODN(A), irradiation at 366 nm; Lane 5, B-ORN(G) +ODN(A), irradiation at 366 nm; Lane 6, B-ORN(C) + ODN(A),irradiation at 366 nm.
150This journal is © The Royal Society of Chemistry [year]

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reactivity.12 Thus, the high selectivity of the terminal labelingof RNA would be extremely useful for investigating thespecific RNA sequence.
155

Table 2 Normalized fluorescence intensity for the terminal modified
product of amino-ODN(CVU), correctly base paired to four ORNs thatdiffered in a single nucleotide position.

B-ORN(U)B-ORN(A)
B-ORN(G)B-ORN(C)

ORNa

Fluorescence intensityb

5´-biotin-r(UGUGCU)-3´
5´-biotin-r(UGAGCU)-3´
5´-biotin-r(UGGGCU)-3´
5´-biotin-r(UGCGCU)-3´

1.0 ± 0.20
0.014 ± 0.008
0.014 ± 0.012
0.031 ± 0.020

Underlined characters indicate a mismatched base. bEach experiment was
160 repeated at least three times.

ODN sequences were synthesized by the conventional
phosphoramidite method by using an Applied Biosystems
200 3400
DNA synthesizer. The coupling efficiency wasmonitored with a trityl monitor. The coupling efficiency of
cyanoethylphosphoramidite of CVU was 97% yield. The
coupling time of cyanoethylphosphoramidite of CVU was 999
sec. They were deprotected by incubation with 28% ammonia
205 for 8 h at 55 ˚C and were purified on a Chemcobond 5-ODS-H
column (10 × 150 mm) by reverse phase HPLC; elution waswith 0.05 M ammonium formate containing 3-20% CH3 CN,
linear gradient (30 min) at a flow rate of 3.0 mL/min.
Preparation of ODNs was confirmed by MALDI-TOF-MS
210 analysis.
Photoligation of ODNs as monitored by HPLCaConclusions
In conclusion, we demonstrated the terminal labeling of RNA
through ODN(CVU). When an ODN containing CVU at the 5´
165 terminal site was photoirradiated with an ORN containing apyrimidine base at the 3´ terminal site in the presence oftemplate DNA, efficient terminal labeling was observed.
Furthermore, terminal labeling of RNA showed a high degreeof single nucleotide specificity. Therefore, this system can be
170 widelyused for photochemical post-modification of theterminal site of RNA. This method is useful for incorporating
functional units such as fluorophores and electrochemical
species. This system is expected to allow the detection of
noncoding RNAs and the investigation of RNA structural and
175 functional analyses.

Experimental
General1

H NMR spectra were measured with Varian Gemini 300 (300
MHz) spectrometer. Coupling constant (J value) are reported
180 in hertz. The chemical shifts are expressed in ppm downfieldfrom tetramethylsilane, using residual chloroform (δ = 7.24 in
1
H NMR) and water (δ = 4.65 in 1 H NMR) as an internal
standard. Mass spectra were recorded on a Voyager-DE PROSF, Applied Biosystems. Irradiation was performed by 25 W
185 transilluminator(FUNAKOSHI, TFL-40, 366 nm, 5,700µW/cm 2 ). HPLC was performed on a Cosmosil 5C18AR
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. Kanto Chemical Silica Gel 60 N was used for
190 silica gel column chromatography. Pre-coated TLC plates
Merck silica gel 60 F 254 was used for monitoring reactions.
The reagents for the DNA synthesizer such as A, G, C, T-βcyanoethyl phosphoramidite, and CPG support werepurchased from Glen Research. Calf intestine alkaline
195 phosphatase (AP) was purchased from Promega. Nuclease P1
was purchased from Yamasa.
Preparation of ODNThis journal is © The Royal Society of Chemistry [year]

The reaction mixture (total volume 60 µL) containing
ODN(CVC) and ORN(U) (each 20 µM, strand concn) in thepresence of template ODN(A) (24 µM, strand concn) in 50
215 mM sodium cacodylate buffer (pH 7.0) and 100 mM sodium
chloride was irradiated with a 25 W transilluminator (366 nm)
at 0 °C for 32 min. After irradiation, the progress of thephotoreaction was monitored by HPLC on a Cosmosil
5C18AR column (4.6 × 150 mm, elution with a solvent
220 mixture of 50 mM ammonium formate, pH 7.0, linear gradientover 30 min from 4% to 10% acetonitrile at a flow rate 0.6
mL/min). The yield was calculated based on ORN(U).
Spectroscopic measurements
The absorbance of the hybrid duplexes was monitored at 260
nm from 4.0 to 80 °C with heating rate of 1.0 °C/min using a
JASCO V-550 UV/VIS spectrophotometer or a BECKMANCOULTER DU 800 UV/Visible spectrophotometer. The CD
spectra were measured from 200 to 350 nm in a 0.1 cm path
length cuvette. All spectra of the duplexes (25 µM, strand
230 concn) were measured at 4.0 °C in a buffer containing 50 mM
sodium cacodylate and 100 mM sodium chloride, pH 7.0. CD
spectra were recorded on a JASCO J-720W spectrometer.
225

Immobilization of amino-labeled ODN
The amino-labeled ODN probe containing CVU was diluted to
235 a concentration of 20 µM in 100 mM sodium cacodylate
buffer (pH 7.0). Spotting was accomplished by using 4 µL
aliquots from a standard micropipette. Binding of aminolabeled ODN probe containing CVC to the surface was
performed over a period of 12 h at room temperature inside a
240 desiccator. After probe immobilization, the glass surface wasrinsed with 0.1% SDS and deionized water. The surface wasdeactivated with a solution made of NaBH 4 (3.75 mg), PBS(1.5 mL), and ethanol (375 µL) over a period of 5 min. The
surface was subsequently washed with deionized water, and245 dried.
Terminal labeling of RNA on a DNA chip
A glass chip spotted with a solution (4 µL) made of 2 µM
ODN(A) and biotin-labeled ORN(U) in 50 mM sodium
cacodylate buffer (pH 7.0) and 100 mM sodium chloride was
250 irradiated at 366 nm for 1 h. After the chip had been washedwith deionized water at 98 °C for 5 min, a PBS solution of
streptavidin-Cy3 conjugate (20 µg/mL) was added to the[journal], [year], [vol], 00–00 | 3

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surface, and the chip was washed twice in PBS. Fluorescencemeasurements were performed on a microarray scanner
255 CRBIO II e (Hitachi), which was equipped with a laser withexcitation wavelength of 532 nm.

Acknowledgements
This article is dedicated to Professor Isao Saito on theoccasion of his 65th birthday. This work was supported by a
260 Precursory Research for Embryonic Science and Technology(PRESTO) grant from the Japan Science and Technology
Agency (JST). Partial support by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan is also acknowledged.
265

Notes and references
1

A. P. Silverman and E. T. Kool, Trends Biotechnol., 2005, 23, 225; A.
P. Silverman and E. T. Kool, Chem. Rev., 2006, 106, 3775.
2 M. M. Mhlanga, D. Y. Vargas, C. W. Fung, F. R. Kramer and S.
Tyagi, Nucleic Acids Res., 2005, 33, 1902.
270 3
R. Kawai, M. Kimoto, S. Ikeda, T. Mitsui, M. Endo, S. Yokoyama
and I. Hirao, J. Am. Chem. Soc., 2005, 127, 17286; K. Moriyama, M.
Kimoto, T. Mitsui, S. Yokoyama and I. Hirao, Nucleic Acids Res.,
2005, 33, e129.
4 S. Sando and E. T. Kool, J. Am. Chem. Soc., 2002, 124, 9686; S.
275
Sando H. Abe and E. T. Kool, J. Am. Chem. Soc., 2004, 126, 1081.
5 J. Sekiguchi and S. Shuman, Biochemistry, 1997, 36, 9073.
6 M. Nilsson, G. Barbany, D. Antson, K. Gertow and U. Landegren,Nat. Biotechnol., 2000, 18, 791.
7 S. Carriero and M. J. Damha, J. Org. Chem., 2003, 68, 8328; S.
280
Carriero and M. J. Damha, Nucleic Acids Res., 2003, 31, 6157.
8 K. Fujimoto, S. Matsuda, N. Takahashi and I. Saito, J. Am. Chem.
Soc., 2000, 122, 5646; I. Saito, Y. Miyauchi, Y. Saito and K.
Fujimoto, Tetrahedron Lett., 2005, 46, 97; Y. Yoshimura, Y. Ito andK. Fujimoto, Bioorg. Med. Chem. Lett., 2005, 15, 1299; K. Fujimoto,
285
Y. Yoshimura, T. Ikemoto, A. Nakazawa, M. Hayashi and I. Saito,
Chem. Commun., 2005, 3177; M. Ogino, Y. Yoshimura, A. Nakazawa,
I. Saito and K. Fujimoto, Org. Lett., 2005, 7, 2853; S. Ogasawara andK. Fujimoto, ChemBioChem, 2005, 6, 1756; S. Ogasawara and K.
Fujimoto, Angew. Chem. Int. Ed., 2006, 45, 4512; K. Fujimoto, S.
290
Matsuda, Y. Yoshimura, T. Matsumura, M. Hayashi and I. Saito,
Chem. Commun., 2006, 3223; Y. Yoshimura, D. Okamura, M. Ogino
and K. Fujimoto, Org. Lett., 2006, 8, 5049.
9 Y. Yoshimura, Y. Noguchi, H. Sato and K. Fujimoto, ChemBioChem,
2006, 7, 598.
295 10 S. Nakano, T. Kanzaki and N. Sugimoto, J. Am. Chem. Soc., 2004,
126, 1088.
11 MALDI–TOF–MS: calcd. 3730.34 for ORN(C)^ODN( CVU) [(M +H)+], found 3730.51. The yield was calculated based on ORN(C).
12 K. L. Buchmueller, B. T. Hill, M. S. Platz and K. M. Weeks, J. Am.
300
Chem. Soc., 2003, 125, 10850; K. L. Buchmueller and K. M. Weeks,Biochemistry, 2003, 42, 13869.

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