Transcription of Unnatural Fluorescent Nucleotides and their Application with Graphene Oxide for the Simple and Direct Detection of miRNA

Article abstract of DOI:10.1002/bkcs.11549

In this study we synthesized two differently sized fluorescent RNA nucleotides, rUthioTP and rUpyrTP, and examined their transcription ability using T7 RNA polymerase. The smaller rUthioTP could be incorporated and extended to produce a corresponding RNA sequence, but rUpyrTP could not. We then used this rUthioTP-containing fluorogenic transcription system, in conjunction with graphene oxide(GO), for the detection of miRNA 146a with high sensitivity and selectivity. This combination of a transcribed RNA product and GO is a simple in situ probing system for the detection of miRNA 146a—one that is less time consuming and more cost-effective.

Full text of DOI:10.1002/bkcs.11549

Article
DOI: 10.1002/bkcs.11549
BULLETIN OF THE
B. H. Le and Y. J. Seo
KOREAN CHEMICAL SOCIETY
Transcription of Unnatural Fluorescent Nucleotides and their
Application with Graphene Oxide for the Simple and Direct Detection
of miRNA
Binh Huy Le† and Young Jun Seo†,‡,
*
†Department of Bioactive Material Sciences, Chonbuk National University, Jeonju 561-756, South Korea
‡Department of Chemistry, Chonbuk National University, Jeonju 561-756, South Korea.
*E-mail: yseo@jbnu.ac.kr
Received June 11, 2018, Accepted July 6, 2018
In this study we synthesized two differently sized fluorescent RNA nucleotides, rUthioTP and rUpyrTP,
and examined their transcription ability using T7 RNA polymerase. The smaller rUthioTP could be incor-
porated and extended to produce a corresponding RNA sequence, but rUpyrTP could not. We then used
this rUthioTP-containing fluorogenic transcription system, in conjunction with graphene oxide(GO), for
the detection of miRNA 146a with high sensitivity and selectivity. This combination of a transcribed
RNA product and GO is a simple in situ probing system for the detection of miRNA 146a—one that is
less time consuming and more cost-effective.
Keywords: Probe, Fluorescent nucleotides, Transcription, miRNA, Graphene oxide
Introduction
develop methods for the efficient incorporation and exten-
sion of fluorescent nucleotides into RNA, due to the active
sites of polymerases being very tight.20
MiRNAs are short (19–24 bases) single-stranded RNAs
whose function is related to gene regulation focused on the
untranslated region of mRNA.1,2 Recently, many diverse
diagnostic tools have been developed for the detection of
miRNA as biomarkers, using different types of oligonucleo-
tide probing systems, including Taqman probes,3 molecular
beacons (MBs),4,5 and nanoparticle-attached oligonucleotide
detection systems.6–10 Most of these probing systems have
employed oligonucleotides, modified with fluorophores and
quenchers, that were prepared using solid phase synthetic
methods. To obtain efficient versions of such probing sys-
tems, the oligonucleotides must be designed carefully to
ensure secondary structures that align the fluorophore and
quencher units appropriately.11–17 Accordingly, the syn-
thetic procedures for preparing oligonucleotide-based prob-
ing systems can be very complex. They can require several
synthetic and purification steps, as well as complex diagnos-
tic procedures in combination with other systems for the
detection of miRNA; such processes can be time-consuming
and expensive.18,19
To develop a simpler, rapid, and direct in situ diagnostic
tool, we wished to apply in vitro transcription, a direct
enzymatic oligonucleotide synthetic method mediated by
T7 RNA polymerase, to incorporate and extend unnatural
fluorescent nucleotides into RNA, and then use the tran-
scribed fluorescent oligonucleotides, in conjunction with
graphene oxide (GO), for the detection of miRNA.
Many different types of unnatural fluorescent nucleotides
have been developed for the labeling of DNA or RNA for a
variety of purposes. Nevertheless, it remains difficult to
In this study we prepared unnatural fluorescent nucleo-
tides based on deoxyuridine modified at the 5-position,
which is tolerated by the active site of polymerases. We
then employed T7 RNA polymerase to produce RNA oligo-
nucleotides containing the fluorescent nucleotides, through
in vitro transcription, because the transcribed RNA could
be isolated from the DNA template and used directly for
the detection of miRNA.
To develop the fluorescent nucleotides, we chose a pyrene
fluorophore,21–24 which is designed from our lab, displaying
strong fluorescence and specific excimer behavior when
aggregated, as well as a thiophene fluorophore, which is
smallest fluorescent material, reported from Tor group.24,25
We attached these fluorescent substrates at the 50-position of
deoxyuridine through Suzuki coupling and then prepared
their corresponding fluorescent nucleotide triphosphates,
rUpyrTP and rUthioTP, through the Yoshiuki method.26,27
NMR spectroscopy and mass spectrometry confirmed the
structures of these compounds (see Appendix S1, Support-
ing Information).
Experimental
General Procedure for Triphosphate Synthesis. Proton
Sponge (1.5 equiv.) and the free nucleoside (1 equiv.) were
dissolved in trimethylphosphate (0.3 M) and cooled to
ꢀ
−20 C. POCl3 (1.5 equiv.) was added dropwise, and then
ꢀ
the purple slurry was stirred at −20 C for 2 h. Tributyla-
mine (6.2 equiv.) was added, followed by a solution of
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
1

Article
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
Table 1. Oligonucleotide sequences investigated in this study.
tributylammonium pyrophosphate (5.0 equiv.) in DMF
(0.5 M). After 5 min, the reaction was quenched through
the addition of 0.5 M aqueous Et3NH2CO3 (20 vol equiv.)
and then the resulting solution was lyophilized. Purification
through reverse-phase (C18) HPLC (4–35% CH3CN in
0.1 M Et3NH2CO3, pH 7.5), followed by lyophilization,
afforded the triphosphate as a solid.
Name
Sequence
ODN 1
ODN 2
50- GCC ATG GGG CTG ATC
50- TGA GAA CTG AAT TCC ATG GGT
TGA TCA GCC CCA TGG C
miRNA 146a
miRNA 21
miRNA 24-3P
miRNA25b
50- uga gaa cug aau ucc aug ggu u
50- uag cuu auc aga cug aug uug a
50- ugg cuc agu uca gca gga aca g
50- ucc cug aga ccc uaa cuu gug a
Transcription Reaction. ODN 1 ꢀand ODN 2 were
annealed (0.2 μM) by heating to 95 C and then cooling
slowly to room temperature. The rNTPs, rUthioTP, and
rUpyrTP (5 mM) were added to samples, which contained
1× DTT and 1× T7 RNA polymerase buffer. All samples
polyaromatic group that presumably did not fit in the
active site.
ꢀ
were incubated at 37 C for 5 h.
Preparation of transcribed RNA interacted with GO.
An appropriate amount of GO solution 0.2 μg (200 μL)
was added into the tube containing C-miRNA 146a
(0.1 μM) in 25 mM Tris–HCl buffer in DEPC water. The
mixture was then vortexed for 2 min. To ensure that the C-
miRNA 146a was completely absorbed on the surface ꢀof
the GO, the solution was centrifuged (13 000 rpm) at 4 C
for 30 min. The liquid phase was collected, and its fluores-
cence emission spectra measured. In other tube, the same
amount of C-miRNA 146a (0.1 μM) was prepared in the
same buffer condition, then GO 0.2 μg (200 μL) was added
into the tube and vortexed for 2 min. After that, the target
miRNA (0.1 μM and 1.5 μM) was added to this solution,
then incubated at room temperature for 10 min. Next, the
mixture was centrifuged (13 000 rpm) at room temperature
for 20 min, the solid phase was removed, and the liquid
phase was collected to measure fluorescence emission
spectra.
After confirming that rUthioTP could be transcribed
well using T7 RNA polymerase, we applied its transcribed
fluorescent RNA product (C-miRNA 146a), in conjunction
with GO as a quencher, for the detection of miRNA. From
experiments in which we varied the ratio of the transcribed
fluorescent RNA to GO (Figure 2), we found that 200 ng
of GO and 0.1 μM of the RNA product provided the opti-
mal quenching state.
We chose miRNA 146a, which is related to human can-
cer29and a Staphylococcus aureus enterotoxin for cows,30
as the target miRNA for detection. When we added
miRNA 146a into the quenched RNA/GO probe system at
a ratio (C-miRNA 146a to miRNA 146a) of 1:1 we did
not observe any significant increase in fluorescence. In con-
trast, when we increased the content of miRNA 146a up to
1:15, the fluorescence increased dramatically (Figure 3),
Sample Preparation for Fluorescence and UV Spectros-
copy. The fluoꢀrescence emission spectra of the RNAs were
recorded at 25 C with excitation at 354 nm using a quartz
cuvette (path length: 1 cm) on a JASCO PF-6500 spectro-
fluorometer (Tokyo 193-0835, Japan). The UV spectra
were recorded using a Cary Series UV–Vis spectrophotom-
eter Agilent (Santa Clara, CA 95051, USA) and a quartz
cell (path length: 1 cm).
Transcribed
product
Transcribed fluorescent product
Results and Discussion
Figure 1. Denaturing polyacrylamide gel data, confirming the
transcription reaction of rUthiTP. Lane 1: miRNA 146a (22mer);
lane 2: ODN 1; lane 3: ODN 2; lane 4: ODN 1 + ODN 2 + T7
RNA polymerase with rNTPs; lane 5: ODN 1 + ODN 2 + T7
RNA polymerase with rNTPs, treated with DNase I; lane 6: ODN
1 + ODN 2 + T7 RNA polymerase with rUthiTP, rCTP, rATP,
and rGTP; lane 7: ODN 1 + ODN 2 + T7 RNA polymerase with
rUthiTP, rCTP, rATP, and rGTP, treated with DNase I; lane 8:
ODN 1 + ODN 2 + T7 RNA polymerase with rUpyrTP, rCTP,
rATP, and rGTP; lane 9: ODN 1 + ODN 2 + T7 RNA polymerase
with rUpyrTP, rCTP, rATP, and rGTP, treated with DNase
For in vitro transcription of rUpyrTP and rUthioTP, we
employed ODN 1 and ODN 2 as the primer and template,
respectively, in conjunction with T7 RNA polymerase to
produce fluorescent RNA oligonucleotide (Table 1).28
Monitoring the transcription reactions through gel electro-
phoresis revealed (Figure 1) that rUthioTP was incorpo-
rated and extended well into the RNA, but rUpyrTP was
not. The successful preparation of the transcribed fluores-
cent RNA was confirmed using DNase I to remove the
DNA primer and template. We suspect that the small size
of the fluorescent unit in rUthioTP resulted in less of a ste-
ric clash with the active site of T7 RNA polymerase, allow-
ing its efficient incorporation and extension during
transcription. In contrast, rUpyrTP presented a bulky
ꢀ
I. ODN 1:ODN 2 was annealed by heating at 95 C and then
cooled slowly to room temperature. T7 RNA polymerase (50 U)
ꢀ
and its buffer containing DTT were added and incubated at 37 C
for 5 h. Reactions were quenched by adding 2 μL of 0.5 M
EDTA. All transcription products were stained after gel electro-
phoresis using the ethidium bromide (EtBr) solution.
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
2

BULLETIN OF THE
Article
Unnatural fluorescent nucleotides for the detection of miRNA
KOREAN CHEMICAL SOCIETY
C-miRNA 146a
C-miRNA 146a + GO
C-miRNA 146a + GO + miRNA 21
C-miRNA 146a + GO + miRNA 24-3P
C-miRNA 146a + GO + miRNA 25b
C-miRNA 146a + GO + target miRNA 146a
C-miRNA 146a
C-miRNA 146a + 10 ng GO
C-miRNA 146a + 20 ng GO
C-miRNA 146a + 40 ng GO
C-miRNA 146a + 60 ng GO
800
800
600
600
400
200
0
C-miRNA 146a + 80 ng GO
C-miRNA 146a + 100 ng GO
C-miRNA 146a + 150 ng GO
400
C-miRNA 146a + 200 ng GO
200
0
450
500
550
600
Wavelength (nm)
Figure 4. Fluorescence spectra recorded to examine the selectivity
of the C-miRNA 146a probe. All samples contained 0.1 μM
(1 mL) of C-miRNA 146a in 20 mM Tris–HCl as well as 200 ng
of GO. C-miRNA 146a and the target (miRNA 146a) and mis-
matched (miRNA21, miRNA24-3P, miRNA 25b) miRNAs were
added at a ratio of 1:15. All samples were excited at 354 nm.
500
600
Wavelength (nm)
Figure 2. Fluorescence spectra recorded to optimize the quench-
ing state formed between C-miRNA 146a and GO. All RNA solu-
tions comprised, 0.1 μM (1 mL) of the RNA product in 20 mM
Tris–HCl and various amounts of GO (10–200 ng). All samples
were excited at 354 nm.
transcribed RNA/GO-based probing system could be used
to detect the target miRNA 146a selectively.
presumably because of displacement of the RNA oligonu-
cleotide probe by the target miRNA 146a from the surface
of the GO and subsequent formation of a duplex between
the probe and the target RNA.31 In this regard, we suspect
that the RNA/GO probing system has less significant sensi-
tivity relative to other probing system.
To examine the selectivity of this probe system, we
tested the effects of various other miRNA sequences. A
high increase in fluorescence occurred only in the presence
of miRNA 146a; we could discriminate the target miRNA
146a from other random mismatched miRNAs which show
low fluorescence intensity relatively (Figure 4). Thus, our
Conclusion
We have synthesized the unnatural fluorescent nucleotides
rUthioTP and rUpyrTP and examined their direct incor-
poration and extension into RNA through transcription
using T7 RNA polymerase. Although rUthioTP could be
incorporated and extended into the RNA, rUpyrTP could
not, presumably because its greater bulk clashed sterically
with the active site of the polymerase. We used the tran-
scribed product containing rUthioTP, in conjunction with
GO, for the detection of miRNA 146a. Because the fluores-
cence of the transcribed product containing rUthioTP was
quenched dramatically in the presence of GO, we used this
system for the direct in situ detection of miRNA 146a. We
observed a change in fluorescence with a high discrimina-
tion factor when the ratio of the concentrations of the
C-miRNA 146a
C-miRNA 146a + GO
800
C-miRNA 146a + GO + miRNA 146a (ratio 1:1)
C-miRNA 146a + GO + miRNA 146a (ratio 1:2)
C-miRNA 146a + GO + miRNA 146a (ratio 1:5)
600
C-miRNA 146a + GO + miRNA 146a (ratio 1:10)
C-miRNA 146a + GO + miRNA 146a (ratio 1:15)
400
GO
200
0
T7 RNA
Pol
5’
3’
C-miRNA 146a
Fluorescence ON
450
500
550
600
Target miRNA 146a
Wavelength (nm)
Figure 3. Fluorescence spectra recorded to examine the sensitivity
of C-miRNA 146a as a probe. All samples contained 0.1 μM
(1 mL) of C-miRNA 146a in 20 mM Tris–HCl as well as 200 ng
of GO. The ratio of C-miRNA 146a to the target miRNA 146a
was increased from 1:1 to 1:15. All samples were excited at
354 nm.
Displacement
Fluorescence OFF
Scheme 1. Schematic representation of the transcription of the
fluorescent nucleotides rUthioTP and rUpyrTP into C-miRNA
146a and its direct application, with GO, in the detection of
miRNA 146a.
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
3

BULLETIN OF THE
Article
Unnatural fluorescent nucleotides for the detection of miRNA
KOREAN CHEMICAL SOCIETY
11. R. A. Cardullo, S. Agrawal, C. Flores, P. C. Zamecnik,
D. E. Wolf, Proc. Natl. Acad. Sci. USA 1988, 85, 8790.
12. S. A. E. Marras, S. Tyagi, F. R. Kramer, Clin. Chim. Acta
2006, 363, 48.
13. G. T. Hwang, Y. J. Seo, B. H. Kim, J. Am. Chem. Soc. 2004,
126, 6528.
14. L. Wang, C. J. Yang, D. Colin, C. D. Medley, S. A. Benner,
W. Tan, J. Am. Chem. Soc. 2005, 127, 15664.
15. P. Conlon, C. J. Yang, Y. Wu, Y. Chen, K. Martinez,
Y. Kim, N. Stevens, A. A. Marti, A. S. Jockusch, N. J. Turro,
W. Tan, J. Am. Chem. Soc. 2008, 130, 336.
16. H. S. Joshi, H. Y. Tor, Chem. Commun. 2001, 6, 549.
17. M. J. Gait, Oligonucleotide Synthesis: A Practical Approach,
IRL Press, Washington, DC, 1984, p. 1.
transcribed RNA product and miRNA 146a was 1:15, pre-
sumably because miRNA 146a displaced the transcribed
RNA product from the surface of the GO. This probing sys-
tem displayed high selectivity, discriminating miRNA 146a
from other random mismatched sequences. Thus, this tran-
scribed RNA product combined with GO is a simple in situ
probing system for the detection of miRNA 146a—in a
less time consuming and more cost-effective manner.
Acknowledgments. This study was supported by the Basic
Science Research Program through the National Research
Foundation of Korea (NRF)(2017R1A2B4002398).
18. I. S. Kim, Y. J. Seo, Bioorg. Med. Chem. Lett. 2014,
24, 1589.
19. A. D. Maria, M. M. Javier, M. I. Adolfo, Front. Chem. 2016,
4, 18.
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
20. D. G. Lee, I. S. Kim, J. W. Park, Y. J. Seo, Chem. Commun.
2014, 50, 7273.
References
21. H. N. Joo, Y. J. Seo, Chem. Commun. 2015, 51, 2939.
22. I. S. Kim, Y. J. Seo, Tetrahedron Lett. 2014, 55, 1461.
23. H. N. Joo, Y. J. Seo, Bioorg. Med. Chem. Lett. 2014,
25, 5286.
24. M. S. Noé, R. W. Sinkeldam, Y. Tor, J. Org. Chem. 2013,
78, 8123.
25. S. G. Srivatsan, Y. Tor, Chem. Asian. J. 2009, 4, 419.
26. B. H. Le, J. C. Koo, H. N. Joo, Y. J. Seo, Bioorg. Med.
Chem. 2017, 25, 3591.
27. H. Marcel, Molecules 2012, 17, 13569.
28. J. S. Thomas, A. Irina, Nat. Rev. Microbiol. 2011, 9, 319.
29. L. Li, X. P. Chen, Y. L. Li, Scand. J. Immunol. 2010,
71, 227.
1. V. Ambros, Cell 2001, 107, 823.
2. D. P. Bartel, Cell 2004, 116, 281.
3. P. M. Holland, R. D. Abramson, R. Watson, D. H. Gelfand,
Proc. Natl. Acad. Sci. USA 1991, 88, 7276.
4. S. Yagi, F. R. Kramer, Nat. Biotechnol. 1996, 14, 303.
5. C. Hong, A. Baek, S. S. Hah, W. Jung, D. E. Kim, Anal.
Chem. 2016, 88, 2999.
6. P. Miao, B. Wang, F. Meng, J. Yin, Y. Tang, Bioconjugate
Chem. 2015, 26, 602.
7. X. Zhang, H. Liu, R. Li, N. Zhang, Y. Xiong, S. Niu, Chem.
Commun. 2015, 51, 6952.
8. J. Li, T. Deng, X. Chu, R. Yang, J. Jiang, G. Shen, R. Yu,
Anal. Chem. 2010, 82, 2811.
9. W. Xu, X. Xie, D. Li, Z. Yang, T. Li, X. Liu, Small 2012,
8, 1846.
30. F. Dilda, G. Gioia, L. Pisani, L. Restelli, C. Lecchi,
F. Albonico, V. Bronzo, M. Mortarino, F. Ceciliani, Vet. J.
2012, 192, 514.
10. S. Tyagi, D. Bratu, F. R. Kramer, Nat. Biotechnol. 1998,
16, 49.
31. B. Liu, Z. Sun, X. Zhang, J. Liu, Anal. Chem. 2013,
85, 7987.
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
4