Site-specific fluorescent labeling of RNA molecules by specific transcription using unnatural base pairs

Article abstract of DOI:10.1021/ja0542946

Site-specific fluorescent labeling of RNA molecules was achieved by specific transcription using an unnatural base pair system. The unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo-(1H)pyridine (y), and 2-amino-6-(2-thiazolyl)purine

Full text of DOI:10.1021/ja0542946

Published on Web 11/12/2005
Site-Specific Fluorescent Labeling of RNA Molecules by
Specific Transcription Using Unnatural Base Pairs
Rie Kawai,† Michiko Kimoto,† Shuji Ikeda,‡ Tsuneo Mitsui,‡ Masayuki Endo,†
Shigeyuki Yokoyama,*,†,§, and Ichiro Hirao*,†,‡
Contribution from the Protein Research Group, RIKEN Genomic Sciences Center,
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan, Research Center
for AdVanced Science and Technology, The UniVersity of Tokyo, 4-6-1 Komaba, Meguro-ku,
Tokyo 153-8904, Japan, Department of Biophysics and Biochemistry, Graduate School of
Science, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and
RIKEN Harima Institute at SPring-8, 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
Received June 29, 2005; E-mail: ihirao@riken.jp; yokoyama@biochem.s.u-tokyo.ac.jp
Abstract: Site-specific fluorescent labeling of RNA molecules was achieved by specific transcription using
an unnatural base pair system. The unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo-
(1H)pyridine (y), and 2-amino-6-(2-thiazolyl)purine (v) and y function in transcription, and the substrates of
y and 5-modified y bases can be site-specifically incorporated into RNA, opposite s or v in DNA templates,
by T7 RNA polymerase. Ribonucleoside 5′-triphosphates of 5-fluorophore-linked y bases were chemically
synthesized from the nucleoside of y. These fluorescent substrates were site-specifically incorporated into
RNA by transcription mediated by the s-y and v-y pairs. By using this fluorescent labeling method, specific
positions of Raf-binding and theophylline-binding RNA aptamers were fluorescently labeled, and the specific
binding to their target molecules was detected by their fluorescent intensities. This site-specific labeling
method using an unnatural base pair system will be useful for analyzing conformational changes of RNA
molecules and for detecting interactions between RNA and its binding species.
Introduction
fragments but becomes cumbersome for RNAs consisting of
more than ∼70 nucleotides. Thus, the direct incorporation of a
Fluorescent labeling of RNA molecules is an important
technique in both basic and applied sciences. Fluorescence-based
detection offers flexible availability, not only for the replacement
of radiolabeled detection, but also for a wide range of RNA-
based biotechnologies,1 such as single-molecule imaging tech-
niques, probes for structural and functional analyses, and
molecular beacons using fluorescence resonance energy transfer
(FRET). For example, structural analyses of dynamic rearrange-
ments of RNA molecules were performed by the site-specific
attachment of fluorophores to RNA.2 Thus, the site-specific
incorporation of fluorophores into RNA at desired positions has
become a powerful method to expand the utility of fluorescently
labeled RNA molecules.
fluorescent agent into RNA during transcription using DNA tem-
plates is a more useful method. The 5′-termini of RNA can be
site-specifically labeled by conventional T7 transcription using
fluorescent cap analogues, such as a coumarin-derivatized
GTP,3,4 or by T7 transcription under the T7 2.5 promoter using
N6- or 5′-fluorescein-derivatized AMP.5 Modified UTP and CTP
that link with fluorophores at position 5 are also available,6 but
these substrates are randomly incorporated into RNA and cannot
be used extensively. Consequently, there have been only a few
reports on fluorescent analogues introduced into large RNA
molecules.
(2) (a) Tuschl, T.; Gohlke, C.; Jovin, T. M.; Westhof, E.; Eckstein, F. Science
1994, 266, 785-789. (b) Jhaveri, S. D.; Kirby, R.; Conrad, R.; Maglott, E.
J.; Bowser, M.; Kennedy, R. T.; Glick, G.; Ellington, A. D. J. Am. Chem.
Soc. 2000, 122, 2469-2473. (c) Jucker, F. M.; Phillips, R. M.; McCallum,
S. A.; Pardi, A. Biochemistry 2003, 42, 2560-2567. (d) Xie, Z.; Srividya,
N.; Sosnick, T. R.; Pan, T.; Scherer, N. F. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 534-539. (e) Blanchard S. C.; Kim, H. D.; Gonzalez, R. L.;
Puglisi, J. D.; Chu, S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12893-
12898. (f) Blanchard, S. C.; Gonzalez, R. L.; Kim, H. D.; Chu, S.; Puglisi,
J. D. Nat. Struct. Mol. Biol. 2004, 11, 1008-1014. (g) Dorywalska, M.;
Blanchard, S. C.; Gonzalez, R. L.; Kim, H. D.; Chu, S.; Puglisi, J. D.
Nucleic Acids Res. 2005, 33, 182-189.
(3) (a) Qin, P. Z.; Pyle, A. M. Methods 1999, 18, 60-70. (b) Cremo, C. R.
Methods Enzymol. 2003, 360, 128-177.
(4) Collett, J. R.; Cho, E. J.; Lee, J. F.; Levy, M.; Hood, A. J.; Wan, C.;
Ellington, A. D. Anal. Biochem. 2005, 338, 113-123.
(5) Huang, F.; Wang, G.; Coleman, T.; Li, N. RNA 2003, 9, 1562-1570.
(6) (a) Langer, P. R.; Waldrop, A. A.; Ward, D. C. Proc. Natl. Acad. Sci.
U.S.A. 1981, 78, 6633-6637. (b) Waggoner, A. Methods Enzymol. 1995,
246, 362-373. (c) ’t Hoen, P. A. C.; de Kort, F.; van Ommen, G. J.; den
Dunnen, J. T. Nucleic Acids Res. 2003, 31, e20. (d) Alpha-Bazin, B.; Bazin,
H.; Boissy, L.; Mathis, G. Anal. Biochem. 2000, 286, 17-25.
The current methods for the site-specific fluorescent labeling
of RNA are still restrictive. One reliable method is the intro-
duction of a fluorescent unit into RNA fragments during chem-
ical RNA synthesis.1a,2a,3 This method is useful for short RNA
† RIKEN Genomic Sciences Center.
‡ Research Center for Advanced Science and Technology, The University
of Tokyo.
§ Department of Biophysics and Biochemistry, The University of Tokyo.
RIKEN Harima Institute.
(1) (a) Walter, N. G.; Burke, J. M. Methods Enzymol. 2000, 317, 409-440.
(b) Yamamoto, R.; Kumar, P. K. R. Genes Cells 2000, 5, 389-396. (c)
Klostermeier, D.; Millar, D. P. Methods 2001, 23, 240-254. (d) Tan, W.;
Wang, K.; Drake, T. J. Curr. Opin. Chem. Biol. 2004, 8, 547-553. (e)
Zhuang, X. Annu. ReV. Biophys. Biomol. Struct. 2005, 34, 399-414. (f)
Mhlanga, M. M.; Vargas, D. Y.; Fung, C. W.; Kramer, F. R.; Tyagi, S.
Nucleic Acids Res. 2005, 33, 1902-1912.
9
17286
J. AM. CHEM. SOC. 2005, 127, 17286-17295
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RNA Fluorescent Labeling by Specific Transcription
A R T I C L E S
Figure 1. The unnatural s-y and v-y base pairs (a) and the nucleoside 5′-triphosphates of the 5-fluorophore-linked y bases (b).
Another attractive approach is to incorporate fluorescent
residues into RNA at desired positions during transcription
mediated by extra base pairs. Recently, we developed unnatural
base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo-
(1H)pyridine (y),7 and 2-amino-6-(2-thiazolyl)purine (v) and y8
(Figure 1a), which exhibit high specificity in transcription. The
bulky 6-(2-thienyl) group of s and the 6-(2-thiazolyl) group of
v prevent noncognate pairing with C or T, but the relatively
small hydrogen at position 6 of y maintains the shape comple-
mentarity of the s-y and v-y pairings. Thus, the substrates of
y and 5-modified y bases can be site-specifically incorporated
into RNA, opposite s or v in DNA templates, by T7 transcrip-
tion.9 In particular, the v-y pair exhibits high efficiency and
selectivity and is suitable for the incorporation of modified y
substrates linked with a large functional group at position 5.9c
Here, we report the syntheses of ribonucleoside 5′-triphos-
phates of 5-fluorophore-linked y bases (Figure 1b) and their
(9) (a) Kimoto, M.; Endo, M.; Mitsui, T.; Okuni, T.; Hirao, I., Yokoyama, S.
Chem. Biol. 2004, 11, 47-55. (b) Endo, M.; Mitsui, T.; Okuni, T.; Kimoto,
M.; Hirao, I.; Yokoyama, S. Bioorg. Med. Chem. Lett. 2004, 14, 2593-
2596. (c) Moriyama, K.; Kimoto, M.; Mitsui, T.; Yokoyama, S.; Hirao, I.
Nucleic Acids Res. 2005, 33, e129.
(7) Hirao, I.; et al. Nat. Biotechnol. 2002, 20, 177-182.
(8) Mitsui, T.; Kimoto, M.; Harada, Y.; Yokoyama, S.; Hirao, I. J. Am. Chem.
Soc. 2005, 127, 8652-8658.
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A R T I C L E S
Kawai et al.
Scheme 1. Synthesis of the Nucleoside 5′-Triphosphates of 5-Fluorophore-Linked y Basesa
a Conditions: (a) N-iodosuccinimide, CH3CN, DMF, 80 °C, 2 h; (b) DMTr-Cl, pyridine, room temperature; (c) (i) (CH3CO)2O, pyridine, room temperature,
(ii) C2H5OH, reflux; (d) CuI, Pd[P(C6H5)3]4, DMF, triethylamine, room temperature, then 2,2-dichloro-N-prop-2-ynyl-acetamide; (e) CHCl2COOH, CH2Cl2,
0 °C; (f) 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one/dioxane, tri-n-butylamine, bis(tri-n-butylammonium)pyrophosphate, I2/H2O/pyridine, dioxane, pyridine,
room temperature, then NH4OH; (g) FAM-N-hydroxysuccinimidyl ester/DMF, 0.1 M NaHCO3-Na2CO3 buffer (pH 8.5), room temperature, then NH4OH;
(h) CuI, Pd[P(C6H5)3]4, DMF, triethylamine, room temperature, then N-(2-propynyl)-6-trifluoroacetamidohexanamide; (i) R-N-hydroxysuccinimidyl ester (R
) FAM or TAMRA)/DMF, 0.1 M NaHCO3-Na2CO3 buffer (pH 8.6), room temperature, 3.5 h, then NH4OH or Dansyl-x-N-hydroxysuccinimidyl ester/
DMF, 0.1 M sodium borate buffer (pH 8.5), room temperature, 48 h, then NH4OH.
Table 1. Absorption and Emission Maxima and Quantum Yields
site-specific incorporation into RNA by T7 transcription, using
DNA templates containing s or v. We also describe the
application of this method to the fluorescent labeling of two
different RNA aptamers.
of Nucleoside 5′-Triphosphates of 5-Fluorophore-Linked y Basesa
absorption maxima
(nm)
emission maxima
(nm)
quantum
yield
compounds
FAM-hx-yTP
TAMRA-hx-yTP 553 (ꢀ 85 000)
FAM-yTP
Dansyl-x-yTP
493 (ꢀ 62 000)
522 (excited at 493 nm) 0.55
578 (excited at 553 nm) 0.50
521 (excited at 493 nm) 0.67
Results and Discussion
493 (ꢀ 70 000)
252 (ꢀ 31 400),
319 (ꢀ 10 600)
Ribonucleosides of 5-fluorophore-linked y bases were syn-
thesized via the nucleoside of 5-iodo-y (Scheme 1). Iodination
of the nucleoside of y was performed by using N-iodosuccin-
imide, and the yield (75%) was improved as compared to our
previously reported yield (34%), which was obtained by using
iodine and potassium iodide.9a Then, the 5′- and 2′,3′-hydroxyl
groups of the nucleoside of 5-iodo-y were protected with a
dimethoxytrityl group and acetyl groups, respectively. The fully
protected nucleoside was reacted with 2,2-dichloro-N-prop-2-
ynyl-acetamide or N-(2-propynyl)-6-trifluoroacetamidohexamide
by palladium-catalyzed coupling, using copper(I) iodide and
triethylamine.10 After deprotection of the 5′-dimethoxytrityl
group, both nucleoside derivatives of 5-[3-(2,2-dichloroacet-
amido)-1-propynyl] y and 5-[3-(6-trifluoroacetamidohexana-
mido)-1-propynyl] y were converted to 5′-triphosphates by a
conventional method.11 The acyl protecting groups in these
compounds were fully removed with concentrated ammonia,
and the triphosphate of 5-(3-amino-1-propynyl) y or 5-[3-(6-
aminohexanamido)-1-propynyl] y (NH2-hx-yTP) was obtained.
The triphosphates were treated with the N-hydroxysuccinimidyl
esters of fluorescent derivatives, 5-carboxyfluorescein (FAM),
5-carboxytetramethylrhodamine (TAMRA), and 6-[(5-dimethyl-
aminonaphthalene-1-sulfonyl)amino]hexanoic acid (Dansyl-x).
The products were purified by DEAE Sephadex column
chromatography and C18 reversed-phase HPLC, and the tri-
phosphates of 5-(3-amino-1-propynyl) y linked with FAM
(FAM-yTP) or 5-[3-(6-aminohexanamido)-1-propynyl] y linked
with FAM (FAM-hx-yTP), TAMRA (TAMRA-hx-yTP), or
387, 541 (excited at
0.014
320 nm)
a The data were measured using a 1 µM concentration of the compound
in 10 mM sodium phosphate buffer (pH 7.0) at 25 °C.
Dansyl-x (Dansyl-x-yTP) were each obtained. The structures
of the products were confirmed by 1H and 31P NMR and mass
spectrometry (see the Supporting Information). The molar
absorption coefficients (ꢀ) determined by quantitative analysis
of the phosphorus in the compound,12 the emission maxima,
and the quantum yields of each triphosphate are shown in Table
1 and Figure 1 in the Supporting Information.
The incorporation efficiency and selectivity of these triphos-
phates into RNA were examined by gel electrophoresis of 32P-
labeled transcripts (17-mer) of DNA templates (35-mer) con-
taining v, s, or A at position +13 (Figure 2 and Figure 2 in the
Supporting Information). T7 transcription was performed using
1 mM natural NTPs and fluorophore-linked yTP with [γ-32P]
GTP, at 37 °C for 3 h (Figure 2a). With the incorporation of
the fluorophore-linked y, the full-length transcripts (17-mer)
exhibited large mobility shifts on the gel (Figure 2b, lanes 1, 3,
5, and 7). Furthermore, in the transcription using the template
containing v, no band corresponding to the native 17-mer was
observed (Figure 2b, lane 9), and in the transcription using the
native template with fluorescent-linked yTP, there was no band
corresponding to the transcripts containing fluorophore-linked
y bases (Figure 2b, lanes 2, 4, 6, and 8). These results strongly
suggest that these fluorophore-linked yTPs were not incorporated
(10) Alvarez, A.; Guzman, A.; Ruiz, A.; Velarde, E.; Muchowski, J. M. J. Org.
Chem. 1992, 57, 1653-1656.
(11) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631-635.
(12) Ohtsuki, T.; Kimoto, M.; Ishikawa, M.; Mitsui, T.; Hirao, I.; Yokoyama,
S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4922-4925.
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RNA Fluorescent Labeling by Specific Transcription
A R T I C L E S
Figure 3. Posttranscriptional fluorophore labeling of an RNA fragment
containing NH2-hx-y. A 5′-32P-labeled RNA fragment (17-mer) containing
NH2-hx-y (20 µM) was labeled with 5-carboxyfluorescein N-hydroxysuc-
cinimidyl ester (FAM-SE) or 5-carboxytetramethylrhodamine N-hydroxy-
succinimidyl ester (TAMRA-SE) in 0.1 M sodium tetraborate-HCl buffer
(pH 8.3). The efficiency (%) of the modification was calculated from the
intensities of the bands corresponding to the modified 17-mer and the
original 17-mer.
the efficiency of this internal modification of the fragment was
relatively lower than that of a terminal modification, such as
the N6 modification of a 5′-terminal adenosine of an RNA
fragment,5 the NH2-hx-y site in the 17-mer was completely
modified in the presence of 1000 molar equivalents of TAMRA-
SE (Figure 3, lane 10). The native RNA fragment was not
modified under the same conditions (Figure 3, lanes 6 and 12),
indicating that the NH2-hx-y site was selectively modified with
these reagents.
Figure 2. T7 transcription mediated by the v-y pair. (a) Schemes of the
experiments. (b) Gel electrophoresis of transcripts using the templates (N
) v or A) with the natural NTPs (1 mM) and each of the substrates of
5-fluorophore-linked y (1 mM). Transcripts were labeled with [γ-32P]GTP.
The relative yields (lanes 1-8) of each transcript were determined by
comparison to the yield of the native transcript from the template consisting
of the natural bases (lane 9), and each yield was averaged from three data
sets.
The site-specific fluorescent-labeling of RNA was applied
to the detection of RNA-protein interactions using an anti-
(Raf-1) RNA aptamer, which specifically binds to the human
Raf-1 protein and inhibits the interaction between Raf-1 and
Ras.13 From a vector encoding the original anti-(Raf-1) RNA
aptamer, the template containing v was prepared and amplified
by PCR, using a 3′-primer containing v (Figure 4a). During T7
transcription, FAM-hx-yTP was incorporated at position 90 of
the aptamer (100-mer), and the transcript was purified by gel
electrophoresis. To monitor the binding ability of the aptamer
to the protein, we performed a filter-binding assay with the
fluorescently labeled aptamer and the Ras-binding domain of
Raf-1 (Raf-1 RBD) (Figure 4b). The RNA aptamer (10 nM)
was incubated with various concentrations (47-975 nM) of the
Raf-1 RBD at room temperature for 30 min. The RNA-protein
complex was isolated by filtration on modified nitrocellulose
filters, and the fluorescent intensity on the filter was measured
with a Bio-Rad Molecular Imager FX Pro system (Figure 4b).
Even in the binding with 50 nM Raf-1 protein (2.5 pmol in 50
µL), the FAM-labeled aptamer could be detected, and the
dissociation constant (Kd ) 184 ( 46 nM) was determined
without any activity loss of the aptamer (Figure 4c). Thus, this
site-specific fluorescent labeling of RNA will be useful as a
nonradiolabeling method for the detection of target molecules
bound with fluorescently labeled RNA aptamers.
into RNA opposite the natural bases and were site-specifically
incorporated opposite v in the template. In addition, since the
site-specific incorporation of yTP opposite v or s was confirmed
by a nucleotide-composition analysis of the transcripts,8 the
fluorophore-linked yTPs might also be incorporated at the
desired position. Although truncated transcripts (12-mer) were
observed, the full-length products (17-mer) containing the
fluorophore-linked y transcribed from the v template were
obtained with relatively high yields (48-80% relative to that
of the transcript with natural bases only; Figure 2, lane 9). The
yields of the transcription mediated by the v-y pair were higher
than those mediated by the s-y pair (Figure 2 in the Supporting
Information).
The triphosphate of 3-(6-aminohexanamido)-1-propynyl y
(NH2-hx-yTP) was also site-specifically incorporated into RNA
with a good yield (Figure 2 in the Supporting Information). The
high selectivity of the NH2-hx-y incorporation was confirmed
by the mobility shift of the transcript on the gel (Figure 2 in
the Supporting Information) and by the nucleotide-composition
analysis (Figure 3 in the Supporting Information). This sub-
strate is useful for posttranscriptional modifications at its
incorporation sites in RNA molecules. To test the modification
of the transcripts, the 5′-32P-labeled 17-mer fragment, containing
NH2-hx-y at position 13, was treated with excess amounts of
5-carboxyfluorescein N-hydroxysuccinimidyl ester (FAM-SE)
or 5-carboxytetramethylrhodamine N-hydroxysuccinimidyl ester
(TAMRA-SE) (0-20 mM) in a buffer (pH 8.3) at 37 °C for 12
h, and the products were analyzed on a gel (Figure 3). Although
Next, to examine the potential of this site-specific fluorescent
labeling, we introduced the fluorophore-linked y into another
(13) Kimoto, M.; Shirouzu, M.; Mizutani, S.; Koide, H.; Kaziro, Y.; Hirao, I.;
Yokoyama, S. Eur. J. Biochem. 2002, 269, 697-704.
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A R T I C L E S
Kawai et al.
Figure 4. Site-specific FAM-hx-y labeling of the anti-(Raf-1) RNA aptamer
and the binding of the aptamer to the Raf-1 RBD. (a) Scheme of the site-
specific fluorescent-labeling of the anti-(Raf-1) RNA aptamer and its filter-
binding assay with the Raf-1 RBD. (b) The fluorescent intensity of the
RNA aptamer bound to the Raf-1 RBD on the filters. A Bio-Rad Molecular
Imager FX Pro system was used to excite the samples at 488 nm and to
detect the fluorescence at 530 nm. (c) Binding curves for the RNA aptamer
with the Raf-1 RBD.
Figure 5. Structure of the theophylline-binding RNA aptamer: the
secondary structure (a), the structural interactions in the binding site (b),
the U6-U23-A28 base triplet (c), the y6-U23-A28 base triplet (d), and the
5-propynyl-y base superposition at the U6 position in the aptamer-
theophylline complex (PDB 1O15) (ref 14f) (e). The 5-propynyl-y base is
shown in red, the theophylline is in blue, and the U6, C22, U23, U24, and
A28 nucleotides are in cyan.
RNA aptamer that specifically binds to the bronchodilator
theophylline2c,14 (Figure 5a). Fluorophores at a specific position
in RNA molecules are useful for detecting conformational
changes by measuring the alterations in the fluorescent intensity,
as well as for sensing target molecules. Theophylline binding
causes a unique structural motif (Figure 5b) to appear in the
RNA aptamer. In the complex, two base triplets, U6-U23-A28
(Figure 5c) and A7-C8-G26, and two independent bases, C22
and U24, surround the theophylline and form a highly specific
ligand-binding site, but this site is not stably formed in the
absence of the ligand.2c,14 Jucker et al. introduced a fluorescent
adenine analogue, 2-aminopurine, into position 27 of the aptamer
to analyze the theophylline binding by stopped-flow fluorescence
spectroscopy.2c Thus, we sought an appropriate position that
could be replaced with fluorophore-linked y, and the theophyl-
line binding was monitored by the change in the fluorescence
intensity.
To determine the appropriate site in the theophylline-binding
aptamer for the introduction of fluorophore-linked y, we first
examined the effects of an unmodified y substitution for U at
position 6, 23, or 24 in the binding motif of the aptamer. Each
aptamer (41-mer) containing y was prepared by T7 transcription
using DNA templates containing s and yTP (Figure 4 in the
Supporting Information). The site-specific incorporation of y
into the aptamers was confirmed by a nucleotide-composition
analysis using 2D TLC (Figure 4 in the Supporting Information).
The dissociation constants (Kd) of the aptamer binding to
theophylline (Table 2 and Figure 5 in the Supporting Informa-
tion) were determined by a published equilibrium method,14a
3
using H-labeled theophylline in a buffer containing 100 mM
HEPES (pH 7.3), 50 mM NaCl, and 5 mM MgCl2. The y
substitution at U23 or U24 significantly reduced the binding
abilities of the aptamers (Kd > 1500 nM), as compared with
that of the original aptamer (Kd ) 331 ( 16 nM). As for the
replacement of U23, the 4-keto group of U23 forms hydrogen
bonds with both U6 and A28 in the base triplet. Since the
(14) (a) Jenison, R. D.; Gill, S. C.; Pardi, A.; Polisky, B. Science 1994, 263,
1425-1429. (b) Zimmermann, G. R.; Jenison, R. D.; Wick, C. L.; Simorre,
J.-P.; Pardi, A. Nat. Struct. Biol. 1997, 4, 644-649. (c) Zimmermann, G.
R.; Shields, T. P.; Jenison, R. D.; Wick, C. L.; Pardi, A. Biochemistry 1998,
37, 9186-9192. (d) Zimmermann, G. R.; Wick, C. L.; Shields, T. P.;
Jenison, R. D.; Pardi, A. RNA 2000, 6, 659-667. (e) Sibille, N.; Pardi, A.;
Simorre, J.-P.; Blackledge, M. J. Am. Chem. Soc. 2001, 123, 12135-12146.
(f) Clore, G. M.; Kuszewski, J. J. Am. Chem. Soc. 2003, 125, 1518-1525.
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RNA Fluorescent Labeling by Specific Transcription
A R T I C L E S
Table 2. Theophylline Binding of RNA Aptamers Containing
Modified y Bases
fluorescent intensities of the aptamers containing FAM-y,
FAM-hx-y, or TAMRA-hx-y increased depending on the
amount of theophylline and doubled with the addition of satu-
rating concentrations of theophylline (5-20 µM) (Figure 6).
unnatural base
inserted positon
Kd [nM]a
Kf [nM]b
FAM-y
FAM-hx-y
TAMRA-hx-y
Ph-y
I-y
y
y
y
U6
U6
U6
U6
U6
U6
U23
U24
206 ( 16
192 ( 11
∼1000
510 ( 64
566 ( 115
488 ( 107
3
Although the Kd value of the TAMRA-hx-y aptamer to H-
labeled theophylline was very high, an increase in the fluorescent
intensity of the aptamer was observed (Figure 6d). These fluor-
escent intensity alterations might be caused by a conformational
rearrangement of the aptamer upon binding to the target theo-
phylline. In the absence of theophylline, these fluorescent resi-
dues might reside inside the aptamer and stack with the neigh-
boring bases, and thus, the stacking would reduce the fluorescent
intensity. Upon theophylline binding, the fluorescent residues
might move outside and become exposed to the solvent, in-
creasing the fluorescent intensity.
536 ( 42
551 ( 68
217 ( 13
>1500
>1500
331 ( 16
unmodified
a The dissociation constant values for theophylline binding were measured
with equilibrium filtration assays using 3H-labeled theophylline (20 nM)
and various RNA concentrations (25-1500 nM). b The equilibrium constant
values for theophylline binding were determined from the equilibrium
fluorescence of 100 nM RNA containing fluorophore-linked y, obtained at
various theophylline concentrations (0-20 µM).
The equilibrium constant values obtained from the fluorescent
intensity alterations of the aptamers (designated by Kf) (Figure
6b and Table 2) were somewhat different from the dissociation
constant values (Kd) obtained by using 3H-labeled theophylline.
In the case of the aptamers containing FAM-y and FAM-hx-y,
the Kf values were larger than their Kd values. This suggests
that when theophylline, at concentrations around 0.2-0.5 µM,
binds to the aptamer, the fluorophores still stack with the
neighboring bases inside the structure. In contrast, the Kf value
of the aptamer containing TAMRA-hx-y was one-half of its Kd
value. This might be because the sterically hindered dimethyl-
amino group of TAMRA facilitates the movement of the
TAMRA residue to the outside, by the weak theophylline
binding, but also reduces the binding ability. In either case, the
Kf values of the aptamers depend on the environmental changes
of the fluorophore residues induced by a certain concentration
of theophylline, and thus, the values are practically useful for
estimating the detection sensitivity of the aptamer as a sensor.
unnatural y base lacks the 4-keto group of U, the replacement
of U23 with y might be unfavorable for the binding. However,
no interaction of the 4-keto group of U24 was detected in the
NMR analysis,14b,c but the 3-imino group of U24 directly
interacted with the theophylline. This suggests that the 4-keto
group of U24 has an important role in forming the binding motif,
and U24 actually takes part in the metal binding and the U-turn
formation in the aptamer structure.14b,d On the other hand, the
aptamer in which U6 was replaced with y efficiently bound to
theophylline, with a dissociation constant of 217 ( 13 nM. This
indicates that the removal of the U6’s 4-keto group does not
adversely affect the binding ability of the aptamer. Actually,
this 4-keto group is not involved in the U6-U23-A28 base triplet
in the aptamer.14b,c In addition, the introduction of 5-modified
y bases, such as 5-phenylethynyl-2-oxo(1H)pyridine (Ph-y)9b
and 5-iodo-2-oxo(1H)pyridine (I-y),9a into the aptamer in place
of U6 still maintained the aptamer’s binding ability (Kd ) 536
( 42 nM for the Ph-y replacement and Kd ) 551 ( 68 nM for
the I-y replacement, Table 2 and Figure 5 in the Supporting
Information). Interestingly, the Kd value of the aptamer modified
with y at position 6 (Kd ) 217 ( 13 nM) was slightly lower
than that of the unmodified aptamer (Kd ) 331 ( 16 nM). Thus,
the substitution of U6 with y allows the formation of the base
triplet to capture the theophylline (Figure 5d), and the U6
position might be a suitable site for the introduction of a
fluorophore-linked y.
The fluorescently labeled aptamers retained their high speci-
ficity for theophylline. The fluorescent intensities of the aptamers
were not enhanced in the presence of an excess amount (20
µM) of caffeine, which is structurally similar to theophylline
(Figure 6, parts a, c, and d). In particular, no significant intensity
alteration of the aptamer labeled with FAM-y was observed with
the addition of 20 µM caffeine (Figure 6a). The sensitivity of
this method is much higher than those of modular aptamer
sensors and fluorescing molecular switches.15 Although the
introduction of another fluorophore, 2-aminopurine, at position
27 of the theophylline-binding aptamer also produced a useful
probe of the binding,2c the preparation of the aptamer containing
2-aminopurine requires chemical synthesis. Thus, the transcrip-
tion system using the unnatural base pairs is the simplest
approach for preparing RNA molecules containing fluorescent
probes at desired positions, and the aptamers site-specifically
labeled with the fluorophore-linked y bases could selectively
and efficiently detect their target molecules.
We prepared aptamers containing FAM-y, FAM-hx-y, or
TAMRA-hx-y at the U6 position by T7 transcription using the
DNA template containing s and examined the binding of
theophylline. The Kd values of the binding of aptamers contain-
ing FAM-y or FAM-hx-y to 3H-labeled theophylline were
approximately 200 nM (Table 2), which is the same as that of
the unmodified and U6-modified aptamers. In addition, the
modeling of the aptamer14f with U6 replaced by 5-propynyl-y
showed that the propynyl group protrudes from the major groove
of the structure (Figure 5e). These results suggest that the
introduction of the large group at the U6 position did not affect
the binding ability. In contrast, the binding ability of the aptamer
In this study, we have described the site-specific fluorescent
labeling of RNA molecules by transcription using unnatural base
pairs. Several fluorophore-linked yTPs were chemically syn-
thesized from the nucleoside of y. These fluorophore-linked
yTPs can be site-specifically incorporated into RNA molecules
by T7 RNA polymerase, using DNA templates containing s or
3
containing TAMRA-hx-y to H-labeled theophylline was sig-
nificantly reduced (Table 2 and Figure 5 in the Supporting
Information).
We then measured the fluorescent intensities of the aptamers
in the presence or absence of theophylline, using an FP-6500
spectrofluorimeter, by monitoring the fluorescent emission. The
(15) (a) Stojanovic, M. N.; Kolpashchikov, D. M. J. Am. Chem. Soc. 2004, 126,
9266-9270. (b) Frauendorf, C.; Ja¨schke, A. Bioorg. Med. Chem. 2001, 9,
2521-2524.
9
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A R T I C L E S
Kawai et al.
Figure 6. Fluorescence spectra of the theophylline-binding RNA aptamers site-specifically labeled with FAM-y (a), FAM-hx-y (c), or TAMRA-hx-y (d),
in the presence of increasing amounts of theophylline (0-20 µM) or caffeine (20 µM). Fluorescence intensity at 522 nm vs concentration curves for theophylline
binding with the FAM-y-labeled RNA aptamer (b).
v. The RNA aptamers labeled with the fluorophore-linked y
bases at specific positions retained the aptamer’s binding ability,
and the binding to their target molecules was detected by the
fluorescent intensity.
tion of the fluorophore-linked y bases into RNA would be a
powerful tool for the detection of interactions between RNA
molecules and their ligands. Conformational changes are
important for many RNA molecules that bind ligands with high
affinity and specificity, causing alterations of the environments
of some nucleotides at specific sites in the RNA molecules.
These alterations can be detected by the intensity changes of
the fluorophores at the specific sites. In addition, by combining
this method with other 5′- or 3′-labeling methods,3-5 different
fluorophores can be incorporated into the same strand of a RNA
molecule, which would be useful for FRET applications. Thus,
this site-specific labeling method using an unnatural base pair
system will be useful for analyzing the local conformational
changes of RNA molecules and for detecting the interactions
between RNA molecules and binding species.
As shown in the labeling of the anti-(Raf-1) RNA aptamer,
the 3′-regions of RNA aptamers are suitable as labeling sites
with the fluorophore-linked yTPs for the detection of the
aptamers. This is because in the in vitro selection, the 3′-regions
of RNA pools are hybridized with primers during reverse
transcription and PCR, and thus, the 3′-regions of the RNA
aptamers generally do not participate in the binding to their
target molecules. In addition, DNA templates containing the
unnatural s or v bases can be easily prepared from the original
templates by PCR, using a 3′-primer containing v or s (Figure
4a). In this regard, this 3′-labeling method might be effective
in a recently developed in vitro selection method using capillary
electrophoresis and an RNA pool that is labeled with FAM at
the 5′-end.16 This is because the tertiary structures of RNA
aptamers often involve the 5′ region, and thus for the fluorescent
labeling of RNA aptamers, their 3′ regions may be better than
the 5′ regions.
Materials and Methods
General. Reagents and solvents were purchased from standard
suppliers and used without further purification. Reactions were
monitored by thin-layer chromatography (TLC) using 0.25 mm silica
gel 60 plates impregnated with 254 nm fluorescent indicator (Merck).
Furthermore, as shown in the site-specific labeling of the
theophylline-binding RNA aptamer, the site-specific incorpora-
(16) Mendonsa, S. D.; Bowser, M. T. J. Am. Chem. Soc. 2005, 127, 9382-
9383.
9
17292 J. AM. CHEM. SOC. VOL. 127, NO. 49, 2005

RNA Fluorescent Labeling by Specific Transcription
A R T I C L E S
Nucleosides and triphosphates were purified with a Gilson HPLC
system using an analytical column (Synchropak RPP, 250 mm × 4.6
mm, Eichron Technologies) or a preparative C18 column (Waters
solution was stirred at room temperature for 3.5 h. The reaction mixture
was diluted with EtOAc. The solution was washed with H2O and brine,
dried with MgSO4, and evaporated in vacuo. 3-(2,3-Di-O-acetyl-5-O-
dimethoxytrityl-â-D-ribofuranosyl)-5-[3-(2,2-dichloroacetamido)-1-pro-
pynyl]-2-oxo(1H)pyridine (251 mg, 89%) was purified from the residue
by silica gel column chromatography (0.5-3% CH3OH in CH2Cl2).
To a solution of 3-(2,3-di-O-acetyl-5-O-dimethoxytrityl-â-D-ribofura-
nosyl)-5-[3-(2,2-dichloroacetamido)-1-propynyl]-2-oxo(1H)pyridine (251
mg, 323 µmol) in dichloromethane (32 mL) was added dichloroacetic
acid (323 µL, 3.92 mmol), and the mixture was stirred at 0 °C for 15
min. The reaction mixture was poured into a saturated NaHCO3 solution
and was stirred vigorously. The aqueous layer was extracted with
dichloromethane five times. The combined organic layer was dried with
MgSO4 and evaporated in vacuo. The product (136 mg, 88%) was
purified from the residue by silica gel column chromatography (2-
10% CH3OH in CH2Cl2).
1
Microbond Sphere, 150 mm × 19 mm). H (270 and 300 MHz), 13C
(68 MHz), and 31P NMR (109 MHz) spectra were recorded on JEOL
EX270 and Bruker AV300 magnetic resonance spectrometers. High-
resolution mass spectra (HRMS) were recorded on a JEOL HX-110 or
JM-700 mass spectrometer.
Oligodeoxynucleotide Synthesis. The phosphoramidite derivatives
of v and s were prepared as described.7,8 DNA fragments were
synthesized on an Applied Biosystems 392 DNA synthesizer using
standard â-cyanoethyl phosphoramidite chemistry. For the synthesis
of DNA fragments containing v, base-labile protection, using phenoxy-
acetyl for A, p-isopropylphenoxyacetyl for G, and acetyl groups for
C, was used,17 and these phosphoramidites of the natural bases were
purchased from Glen Research (Virginia). In addition, the acetic
anhydride for the capping step was replaced by phenoxyacetic anhydride
in the reagent.18 DNA fragments were purified by gel electrophoresis.
3-(â-D-Ribofuranosyl)-5-iodo-2-oxo(1H)pyridine. 3-(â-D-Ribofura-
nosyl)-2-oxo(1H)pyridine (378 mg, 1.66 mmol) was dissolved in
CH3CN and evaporated to dryness in vacuo twice. The residue was
dissolved in DMF (3.3 mL) and CH3CN (3.3 mL) with N-iodosuccin-
imide (748 mg, 3.32 mmol), and the solution was stirred at 80 °C for
2 h. After cooling, 1 M NaHCO3 (3.3 mL) was added to the reaction
mixture, and it was evaporated in vacuo. The residue was dissolved in
water (17 mL), and then acetic acid (189 µL) and a 5% sodium
hydrogen sulfite solution (5 mL) were added. The product (441 mg,
75%) was purified by reversed-phase HPLC (Waters Microbond Sphere
model C18, with gradients of 4-15% (6 min) and 15-30% (6 min)
CH3CN in water). The NMR spectra of the product were identical to
the published data.9a
3-(5-O-Dimethoxytrityl-â-D-ribofuranosyl)-5-iodo-2-oxo(1H)-
pyridine. 3-(â-D-Ribofuranosyl)-5-iodo-2-oxo(1H)pyridine (178 mg,
0.51 mmol) was coevaporated with dry pyridine three times. The residue
was dissolved in pyridine (5 mL) with 4,4′-dimethoxytrityl chloride
(183 mg, 0.54 mmol), and the solution was stirred at room temperature
for 1.5 h. The solution was partitioned with EtOAc and H2O. The
organic layer was washed with a saturated NaHCO3 solution twice,
dried with MgSO4, and evaporated in vacuo. The residue was purified
by silica gel column chromatography (0-2% CH3OH in CH2Cl2) to
obtain the dimethoxytrityl derivative (272 mg, 82%).
3-(2,3-Di-O-acetyl-5-O-dimethoxytrityl-â-D-ribofuranosyl)-5-iodo-
2-oxo(1H)pyridine. A quantity of 3-(5-O-dimethoxytrityl-â-D-ribo-
furanosyl)-5-iodo-2-oxo(1H)pyridine (266 mg, 0.41 mmol) was co-
evaporated with dry pyridine three times. The residue was dissolved
in pyridine (4 mL) with acetic anhydride (110 µL, 1.17 mmol), and
the solution was stirred overnight at room temperature. The solution
was partitioned with EtOAc and H2O. The organic layer was washed
with a saturated NaHCO3 solution and brine, dried with MgSO4, and
evaporated in vacuo. The residue was dissolved in ethanol (50 mL)
and refluxed for 1.5 h. After evaporation, the crude product was purified
by silica gel column chromatography (1-4% CH3OH in CH2Cl2) to
yield the acetylated derivative (253 mg, 84%).
3-(2,3-Di-O-acetyl-â-D-ribofuranosyl)-5-[3-(2,2-dichloroacetamido)-
1-propynyl]-2-oxo(1H)pyridine. 3-(2,3-Di-O-acetyl-5-O-dimethoxytri-
tyl-â-D-ribofuranosyl)-5-iodo-2-oxo(1H)pyridine (268 mg, 362 µmol)
was dissolved in CH3CN and evaporated to dryness in vacuo twice.
The residue was dissolved in DMF (2 mL) with Cu(I)I (12.4 mg, 65.2
µmol) and Pd[P(C6H5)3]4 (37.7 mg, 32.6 µmol), and after the addition
of triethylamine (91 µL, 652 µmol), it was stirred in the dark at room
temperature. To the solution, 2,2-dichloro-N-prop-2-ynyl-acetamide
(162 mg, 978 µmol) in DMF (1.5 mL) was added dropwise, and the
3-(â-D-Ribofuranosyl)-5-[3-(fluorescein-5-carboxamido)-1-propy-
nyl]-2-oxo(1H)pyridine 5′-triphosphoric acid (FAM-yTP). 3-(2,3-
Di-O-acetyl-â-D-ribofuranosyl)-5-[3-(2,2-dichloroacetamido)-1-propynyl]-
2-oxo(1H)pyridine (47.5 mg, 0.1 mmol) was dissolved in pyridine and
evaporated to dryness in vacuo. The residue was dissolved in pyridine
(100 µL) and dioxane (300 µL). Then, a 1 M solution of 2-chloro-4H-
1,3,2-benzodioxaphosphorin-4-one in dioxane (110 µL, 0.11 mmol) was
added. After 10 min, tri-n-butylamine (100 µL) and 0.5 M bis(tri-n-
butylammonium)pyrophosphate in DMF (300 µL, 0.15 mmol) were
quickly added to the mixture. The reaction mixture was stirred at room
temperature for 10 min. A solution of 1% iodine in pyridine/water (2
mL, 98/2, v/v) was then added. After 15 min, the oxidation was
quenched by the addition of a 5% sodium hydrogen sulfite solution
(150 µL). The reaction mixture was evaporated in vacuo, and the residue
was dissolved in water (10 mL). After 30 min, concentrated ammonia
(20 mL) was added to the solution, and the reaction mixture was stirred
at room temperature for 5 h and lyophilized. The residue was treated
with concentrated ammonia (3 mL) at 55 °C for 3 h to remove the
protecting groups and was concentrated. The residue was purified
by DEAE Sephadex A-25 column chromatography (1.5 cm × 30
cm, eluted by a linear gradient from 50 mM to 1 M TEAB) to give
3-(â-D-ribofuranosyl)-5-(3-amino-1-propynyl)-2-oxo(1H)pyridine 5′-tri-
phosphoric acid. This was dissolved in 0.1 M NaHCO3-Na2CO3 (pH
8.6, 4 mL) and was reacted with 5-carboxyfluorescein N-hydroxysuc-
cinimidyl ester (FAM-SE) (14.5 mg, 30.6 µmol) in DMF (1 mL) in
the dark at room temperature. After 12 h, the reaction mixture was
treated with concentrated ammonia (1 mL) for 30 min. The product
(10 µmol, 10%) was purified by DEAE Sephadex A-25 column
chromatography (1.5 cm × 30 cm, eluted by a linear gradient from 50
mM to 1 M TEAB) and by C18 HPLC (eluted by a linear gradient of
0%-50% CH3CN in 100 mM TEAA, pH 7.0).
3-(2,3-Di-O-acetyl-5-O-dimethoxytrityl-â-D-ribofuranosyl)-5-[3-
(6-trifluoroacetamidohexanamido)-1-propynyl]-2-oxo(1H)pyri-
dine. 3-(2,3-Di-O-acetyl-5-O-dimethoxytrityl-â-D-ribofuranosyl)-5-iodo-
2-oxo(1H)pyridine (244 mg, 330 µmol) was dissolved in CH3CN
and evaporated to dryness in vacuo twice. The residue was dissolved
in DMF (2 mL) with Cu(I)I (13.3 mg, 69.8 µmol) and Pd[P(C6H5)3]4
(36.4 mg, 31.5 µmol), followed by triethylamine (85 µL, 610 µmol),
and was stirred in the dark at room temperature. To the solution,
N-(2-propynyl)-6-trifluoroacetamidohexanamide (240 mg, 908 µmol)
in DMF (1.5 mL) was added dropwise, and the solution was stirred
at room temperature for 3.5 h. The reaction mixture was diluted
with hexane/EtOAc (1:1). The combined solution was washed with
H2O and brine, dried with MgSO4, and evaporated in vacuo. The
product (255 mg, 88%) was purified from the residue by silica
gel column chromatography (0.5-3% CH3OH in CH2Cl2).
3-(2,3-Di-O-acetyl-â-D-ribofuranosyl)-5-[3-(6-trifluoroacetami-
dohexanamido)-1-propynyl]-2-oxo(1H)pyridine. To a solution of
3-(2,3-di-O-acetyl-5-O-dimethoxytrityl-â-D-ribofuranosyl)-5-[3-(6-tri-
fluoroacetamidohexanamido)-1-propynyl]-2-oxo(1H)pyridine (245 mg,
(17) (a) Schulhof, J. C.; Molko, D.; Te´oule, R. Nucleic Acids Res. 1987, 15,
397-416. (b) Kumar, P.; Gupta, K. C. Nucleic Acids Res. 1997, 25, 5127-
5129.
(18) Chaix, C.; Molko, D.; Te´oule, R. Tetrahedron Lett. 1989, 30, 71-74.
9
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A R T I C L E S
Kawai et al.
279 µmol) in dichloromethane (46 mL) was added dichloroacetic acid
(470 µL, 5.69 mmol), and the mixture was stirred at 0 °C for 15 min.
The reaction mixture was poured into a saturated NaHCO3 solution
and was stirred vigorously. The aqueous layer was extracted with
dichloromethane 20 times. The combined organic layer was dried with
MgSO4 and evaporated in vacuo. The product (119 mg, 74%) was
purified from the residue by silica gel column chromatography (0.5-
4% CH3OH in CH2Cl2).
Emission Spectra and Quantum Yields. Fluorescence spectra of
1 µM triphosphate solutions in 10 mM sodium phosphate buffer (pH
7.0) were measured on a JASCO FP-6500ST spectrofluorimeter at 25
°C. The spectra were collected for the instrument response (1 s), and
all slits were set to 3 nm. Fluorescence quantum yields (Φf) were
calculated by the equation Φf ) (FAsη2Fs)/(AFsη0 ), where the subscript
2
s refers to the standard and Φ is the quantum yield, F is the corrected,
integrated fluorescence, A is the absorption at the excitation wavelength,
η is the refractive index of phosphate buffer (1.33), and η0 is the
refractive index of H2O (1.33). Quantum yield standards were
fluorescein (1.0 µM in 0.1 N NaOH, Φs ) 0.90) and quinine sulfate
(10 µM in 1 N H2SO4, Φs ) 0.55).
T7 Transcription. Templates (10 µM) were annealed in 10 mM
Tris-HCl buffer (pH 7.6) containing 10 mM NaCl, by heating at 95
°C for 3 min and slow cooling to 4 °C. Transcription was carried out
in 40 mM Tris-HCl buffer (pH 8.0), 24 mM MgCl2, 2 mM spermidine,
5 mM DTT, 0.01% TritonX-100, 1 mM natural NTPs, 1 mM modified
yTP, 2 µCi [γ-32P]GTP, 2 µM template, and 50 units of T7 RNA
polymerase (Takara, Kyoto). After an incubation at 37 °C for 3 h, the
reaction was quenched by the addition of a dye solution (20 µL)
containing 10 M urea and 0.05% BPB. The mixture was heated at 75
°C for 3 min and was loaded onto a 20% polyacrylamide-7 M urea
gel. The products on the gels were analyzed with a Bio-imaging analyzer
(BAS2500).
3-(â-D-Ribofuranosyl)-5-[3-(6-aminohexanamido)-1-propynyl]-2-
oxo(1H)pyridine 5′-triphosphoric acid (NH2-hx-yTP). 3-(2,3-Di-O-
acetyl-â-D-ribofuranosyl)-5-[3-(6-trifluoroacetamidohexanamido)-1-
propynyl]-2-oxo(1H)pyridine (58.3 mg, 102 µmol) was coevaporated
with dry pyridine three times. The residue was dissolved in pyridine
(100 µL) and dioxane (300 µL). A 1 M solution of 2-chloro-4H-1,3,2-
benzodioxaphosphorin-4-one in dioxane (110 µL, 110 µmol) was added
to the solution, and then the reaction mixture was stirred at room
temperature for 10 min. Tri-n-butylamine (100 µL) was added to the
reaction mixture, followed by a 0.5 M solution of bis(tri-n-butyl-
ammonium)pyrophosphate in DMF (300 µL). After 10 min, a 1%
solution of iodine/H2O/pyridine (2 mL) was added to the reaction
mixture. After stirring for 15 min, the oxidation was quenched by the
addition of a 5% sodium hydrogen sulfite solution (150 µL). Then the
reaction mixture was evaporated in vacuo, and the residue was dissolved
in H2O (10 mL). After 30 min, concentrated ammonia (12 mL) was
added to the solution, and the reaction mixture was stirred at room
temperature for 3 h and lyophilized. The residue was treated with
concentrated ammonia (4 mL) at room temperature for 3 h to remove
the protecting groups and was lyophilized. The residue was purified
by DEAE Sephadex A-25 column chromatography (1.5 cm × 30 cm,
eluted by a linear gradient from 50 mM to 1 M TEAB) and by C18
HPLC (eluted by a linear gradient of 0-15% CH3CN in 100 mM
TEAA, pH 7.0).
Posttranscriptional Fluorophore Labeling of an RNA Fragment
Containing NH2-hx-y. An RNA fragment (17-mer) containing
NH2-hx-y (20 µM, 2.5 µL) was labeled with a DMSO solution of
FAM-SE or TAMRA-SE (0 or 0.2-20 mM, 2.5 µL) in 0.1 M sodium
tetraborate-HCl buffer (pH 8.3) (15 µL). Reactions were incubated at
37 °C for 12 h. The reaction was quenched by the addition of the dye
solution (20 µL). The mixture was heated at 75 °C for 3 min and was
loaded onto a 20% polyacrylamide-7 M urea gel. The products on the
gels were analyzed with the Bio-imaging analyzer.
3-(â-D-Ribofuranosyl)-5-[3-[6-(fluorescein-5-carboxamido)hex-
anamido]-1-propynyl]-2-oxo(1H)pyridine 5′-triphosphoric acid
(FAM-hx-yTP). A 0.1 M NaHCO3-Na2CO3 buffer solution (pH 8.6,
1.5 mL) of NH2-hx-yTP (10 µmol) was reacted with FAM-SE (5.5
mg, 11.6 µmol) in DMF (200 µL) in the dark at room temperature.
After 3.5 h, the reaction mixture was treated with concentrated ammonia
(1 mL) for 2 h. The product (3.0 µmol, 30%) was purified by DEAE
Sephadex A-25 column chromatography (1.5 cm × 30 cm, eluted by
a linear gradient from 50 mM to 1 M TEAB) and by C18 HPLC (eluted
by a linear gradient of 0-50% CH3CN in 100 mM TEAA, pH 7.0).
3-(â-D-Ribofuranosyl)-5-[3-[6-(tetramethylrhodamine-5-carboxa-
mido)hexanamido]-1-propynyl]-2-oxo(1H)pyridine 5′-triphosphoric
acid (TAMRA-hx-yTP). A 0.1 M NaHCO3-Na2CO3 buffer solution
(pH 8.6, 1.2 mL) of NH2-hx-yTP (8 µmol) was reacted with 5-car-
boxytetramethylrhodamine N-hydroxysuccinimidyl ester (TAMRA-SE)
(5 mg, 9.5 µmol) in a DMF/H2O mixture (150/150 µL), in the dark at
room temperature. After 3.5 h, the reaction mixture was treated with
concentrated ammonia (1 mL) for 2 h. The product (4.0 µmol, 50%)
was purified by DEAE Sephadex A-25 column chromatography (1.5
cm × 30 cm, eluted by a linear gradient from 50 mM to 1 M TEAB)
and by C18 HPLC (eluted by a linear gradient of 0%-50% CH3CN in
100 mM TEAA, pH 7.0).
Preparation of a Fluorescently Labeled RNA Aptamer. The DNA
template for the fluorescently labeled anti-(Raf-1) RNA aptamer (RNA
9A)13 at position 90 was amplified from the TOPO-9A vector,13 by
PCR using a 5′-end primer (39.45) and a 3′-end primer containing v
(29.45v90) [39.45, 5′-GGTAATACGACTCACTATAGGGAGTG-
GAGGAATTCATCG; 29.45v90, 5′-GCAGAAGCTTvCTGTCGCT-
AAGGCATATG]. PCR was carried out in 10 mM Tris-HCl buffer
(pH 8.3), with 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 1
µM each primer, 1 ng/µL TOPO-9A, and 0.025 U/µL Taq DNA
polymerase (Takara, Kyoto), on a PTC-100 program thermal controller
(MJ Research, Inc., Waltham, MA). The conditions were 15 cycles of
denaturation at 94 °C for 15 s, primer annealing at 50 °C for 30 s, and
primer extension at 72 °C for 1 min, followed by an incubation at 72
°C for 5 min. After a treatment with Micropure-EZ enzyme removers
(Millipore, Billerica, MA), the PCR product was precipitated with
ethanol, and then resuspended in water. Transcription was carried out
in a buffer (100 µL) containing 40 mM Tris-HCl (pH 8.0), 8 mM
MgCl2, 2 mM spermidine, 5 mM DTT, 0.01% Triton X-100, 1 mM
natural NTPs, 1 mM FAM-yTP, 2.5 µg of the template DNA, and 250
units of T7 RNA polymerase (Takara, Kyoto). After an incubation at
37 °C for 6 h, the reaction was quenched by the addition of the dye
solution (100 µL). The mixture was heated at 75 °C for 3 min, and
then was loaded onto a 10% polyacrylamide-7 M urea gel. The full-
length, fluorescently labeled product was eluted from the gel with water
and was precipitated with ethanol.
3-(â-D-Ribofuranosyl)-5-[3-[6-[6-[(5-dimethylaminonaphthalene-
1-sulfonyl)amino]hexanamido]hexanamido]-1-propynyl]-2-oxo(1H)-
pyridine 5′-triphosphoric acid (Dansyl-x-yTP). A solution of NH2-
hx-yTP (9 µmol) in a 0.1 M sodium borate buffer solution (pH 8.5,
1.8 mL) was reacted with 6-[(5-dimethylaminonaphthalene-1-sulfonyl)-
amino]hexanoic acid N-hydroxysuccinimidyl ester (20 mg, 44 µmol)
in DMF (1.8 mL), in the dark at room temperature. After 48 h, the
reaction mixture was treated with concentrated ammonia (1 mL) for 1
h. The product (6.2 µmol, 68%) was purified by DEAE Sephadex A-25
column chromatography (1.5 cm × 30 cm, eluted by a linear gradient
from 50 mM to 1 M TEAB) and by C18 HPLC (eluted by a linear
gradient of 0-90% CH3CN in 100 mM TEAA, pH 7.0).
Filter-Binding Assay. The FAM-labeled RNA aptamer (10 nM final
concentration) was incubated with various concentrations (47-975 nM)
of the target protein, the Ras-binding domain (RBD) of Raf-1 fused
with glutathione S-transferase (Raf-1 RBD), in 60 µL of binding buffer
(PBS with 5 mM MgCl2, buffer A), for 30 min at room temperature
(22 °C). The solution (50 µL) was then gently vacuum-filtered over a
HAWP filter (HAWP01300, Millipore, Billerica, MA) and was washed
with 200 µL of buffer A. The filters were exposed and the blots were
9
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RNA Fluorescent Labeling by Specific Transcription
A R T I C L E S
quantified using a Bio-Rad Molecular Imager FX Pro system and the
Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA). The
dissociation constant (Kd) was calculated with Kaleidagraph (Abelbeck
Software, Reading, PA), by using the general curve fit for y ) A0A1/
(A0 + A2), where y is the counts per unit area (mm2) on the filter, A0
is the protein concentration, A1 is the expected counts per unit area
(mm2) on the filter when a fully saturated concentration of the protein
is used, and A2 is the Kd.
Kaleidagraph using the following formula: y ) M0M1/(M0 + M2),
where y is the fraction of theophylline binding, M0 is the RNA
concentration, M1 is the binding capacity of theophylline, and M2 is
the Kd.
Fluorescent Measurements. Fluorescent profiles of each theophyl-
line-binding aptamer containing FAM-y or FAM-hx-y or TAMRA-
hx-y at the U6 position, in the presence of theophylline (0-20 µM) or
caffeine (20 µM), were measured using an FP-6500 spectrofluorimeter
(JASCO) at 25 °C. For the emission spectra, a 100 nM concentration
of the aptamer was used, and the excitation wavelength was set at 434
nm (the aptamer containing FAM-y or FAM-hx-y) or at 500 nm (the
aptamer containing TAMRA-hx-y) with a 5-nm spectral bandwidth.
The Kf values, determined from the equilibrium fluorescence of each
modified aptamer, were obtained with the program Kaleidagraph, using
the following formula: y ) 1 + C0C1/(C0 + C2), where y is the relative
fluorescent intensity at 522 nm for FAM and at 578 nm for TAMRA
(the fluorescent intensity in the absence of theophylline was set to 1),
C0 is the theophylline concentration, C1 corresponds to the relative
increment value at the saturating concentration of theophylline, and
C2 is the Kf.
Site-Specific Incorporation of y or Its 5-Modified Bases into the
Theophylline-Binding Aptamer by T7 Transcription. The chemically
synthesized double-stranded DNA templates containing s were used
for the introduction of y or its modified bases (Ph-y, I-y, and
fluorophore-linked y) in place of the uridine at position 6, 23, or 24.
The sequences of the templates containing s are listed in the Supporting
Information. The numbering of the RNA aptamer (41-mer) corresponds
to that for the construct (33-mer).13 Templates (10 µM of a 59-mer
coding DNA and a 59-mer noncoding DNA) were annealed in a buffer
containing 10 mM Tris-HCl (pH 7.6) and 10 mM NaCl, by heating at
95 °C and slow cooling to 4 °C. Transcription was carried out in a
buffer containing 40 mM Tris-HCl (pH 8.0), 24 mM MgCl2, 2 mM
spermidine, 5 mM DTT, 0.01% Triton X-100, 1 mM natural NTPs, 1
mM unmodified yTP, or 1 mM Ph-yTP, or 0.25 mM I-yTP, or 1 mM
fluorophore-linked yTP (FAM-yTP, or FAM-hx-yTP, or TAMRA-hx-
yTP), 2 µM template, and 2.5 units/µL of T7 RNA polymerase (Takara,
Kyoto). After an incubation at 37 °C for 6 h, the reaction was quenched
by adding an equivalent volume of the dye solution. The reaction
mixture was heated at 75 °C for 3 min, and the 41-mer transcripts
were purified by gel electrophoresis.
Equilibrium Filtration Assays. Theophylline binding by the RNA
aptamers containing y or its 5-modified bases at specific positions was
assessed by the equilibrium filtration assay.14a The various concentra-
tions of aptamers (54 µL; final concentration, 25-1500 nM in 100
mM Hepes (pH 7.3), 50 mM NaCl, and 5 mM MgCl2) were mixed
with 6 µL of 200 nM 3H-labeled theophylline (Moravek), and the
mixtures were incubated at 25 °C for 5 min. The mixture was then
placed in a Microcon YM-10 filtration device (Amicon) and was
centrifuged. A 10-µL sample of the filtrate was removed, and the
radioactivity was determined by scintillation counting. The fraction of
the theophylline binding to each RNA aptamer was determined by the
difference between the theophylline concentration in the filtrates
obtained in the presence and absence of the RNA. The dissociation
constant (Kd) for theophylline binding was determined with the program
Acknowledgment. This work was supported by the RIKEN
Structural Genomics/Proteomics Initiative (RSGI), the National
Project on Protein Structural and Functional Analyses, Ministry
of Education, Culture, Sports, Science and Technology of Japan,
and by a Grant-in-Aid for Scientific Research (KAKENHI
15350097) from the Ministry of Education, Culture, Sports,
Science, and Technology. We are grateful to Dr. Makoto
Komiyama, The University of Tokyo, for his support and helpful
discussions.
Supporting Information Available: NMR and MS data for
the nucleoside derivatives of modified y bases; absorption and
emission spectra of each nucleoside 5′-triphosphate of the
5-fluorophore-linked y base; incorporation experiments of
5-modified yTPs by T7 transcription and the nucleotide-
composition analyses of the transcripts; binding curves for the
modified RNA aptamers; complete list of authors for ref 7. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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J. AM. CHEM. SOC. VOL. 127, NO. 49, 2005 17295