Direct incorporation and extension of a fluorescent nucleotide through rolling circle DNA amplification for the detection of microRNA 24-3P

Article abstract of DOI:10.1016/j.bmcl.2018.04.058

We designed and synthesized several fluorescent nucleotides from thiophene, anthracene and pyrene, which have different sizes, and screened their incorporation and extension capability during the rolling circle amplification of DNA. The thiophene-based fluorescent nucleotide (dUthioTP) could highly incorporate and extended into the rolling circle DNA product, while other fluorescent nucleotides (dUanthTP, and dUpyrTP) could not. This dUthioTP fluorescent nucleotide could be used for the detection of miRNA 24-3P, which is related PRRSV. This direct labeling system during rolling circle DNA amplification exhibited an increased fluorescence signal showing gel formation for the detection of miRNA 24-3P. This direct labeling system is a very simple and cost-efficient method for the detection miRNA 24-3P and also exhibited highly sensitive and selective detection properties.

Full text of DOI:10.1016/j.bmcl.2018.04.058

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Direct incorporation and extension of a fluorescent nucleotide through
rolling circle DNA amplification for the detection of microRNA 24-3P
Binh Huy Le a, Young Jun Seo a,b,
⇑
a Department of Bioactive Material Sciences, Research Center of Bioactive Materials Chonbuk National University, Jeonju 561-756, South Korea
b Department of Chemistry, Chonbuk National University, Jeonju 561-756, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized several fluorescent nucleotides from thiophene, anthracene and pyrene,
which have different sizes, and screened their incorporation and extension capability during the rolling
circle amplification of DNA. The thiophene-based fluorescent nucleotide (dUthioTP) could highly incor-
porate and extended into the rolling circle DNA product, while other fluorescent nucleotides (dUanthTP,
and dUpyrTP) could not. This dUthioTP fluorescent nucleotide could be used for the detection of miRNA
24-3P, which is related PRRSV. This direct labeling system during rolling circle DNA amplification
exhibited an increased fluorescence signal showing gel formation for the detection of miRNA 24-3P.
This direct labeling system is a very simple and cost-efficient method for the detection miRNA 24-3P
and also exhibited highly sensitive and selective detection properties.
Received 15 February 2018
Revised 16 April 2018
Accepted 24 April 2018
Available online xxxx
Keywords:
Fluorescent nucleotide
RCA
Direct labeling
miRNA
Ó 2018 Published by Elsevier Ltd.
Virus
MiRNAs are small endogenous single-stranded noncoding RNAs
that regulate gene expression with a focus on the translation.1
There have been many different types of miRNAs reported related
to diverse cell processes, such as cell proliferation, differentiation,
stress resistance, and apoptosis.2,3 This diverse relationship of miR-
NAs in cell processes could allow miRNA to be used as a biomarker,
particularly targeting several human diseases as well as animal
diseases.4–7 miRNA also has a critical key role in viral infections
by regulating the expression of virus RNA or DNA. Porcine repro-
ductive and respiratory syndrome virus (PRRSV) induces a severe
disease in pigs and is one of the most significant viruses that causes
large economic losses.8 miR-24-3P promoted PRRSV replication
and could be used as a biomarker for the detection of PRRSV.9
For the detection of miRNAs, many different types of probing
systems have been developed, such as real time PCR10,11, northern
blot12, and antibody.13 However, for the practical detection of
miRNA, the extremely low number of copies of miRNA in the blood
is limiting and causes low sensitivity. Thus, researchers have tried
to amplify the signal by employing a degradation enzyme such as
exonuclease III14 or duplex specific enzyme.15 The rolling circle
amplification (RCA) method16 is another alternative efficient
method for signal amplification. Based on the rolling circle
amplification system, many different types of visualization
methods have been developed using intercalative dye17, SYBR
Green18, fluorescent primers19, stem-loop molecular beacons20,21
,
a G-quadruplex-gold combination system22, or a graphene oxide
based probing system.23
However, most of these visualization methods are indirect sig-
nal amplification methods and use complicated processes, which
are time-consuming and have a high cost. Our goal, with regards
to that point of view, was to develop a simple direct labeling sys-
tem during the RCA process without further complicated labeling
processes (Scheme 1). For this purpose, we developed a fluorescent
nucleotide triphosphate, which could be incorporated and
extended into the rolling circle amplified DNA showing a fluores-
cence signal increase. For this purpose, the most important process
is to find a fluorescent nucleotide that could be recognized by
Phi29 DNA polymerase and incorporated and extended into the
RCA DNA. This is an exceedingly challenging and promising task
because the active site of DNA polymerase is very tight and restric-
tive against mutated nucleotides.
We screened three different sized fluorophores including pyr-
ene (four-membered aromatic ring), anthracene (three-membered
aromatic ring), and thiophene (one aromatic ring) based on deoxy
uridine.24 Pyrene is a large-sized efficient fluorophore, which
shows unique excimer-fluorescence properties when they organize
together.25 Anthracene is mid-sized fluorophore, which has a light-
induced dimerization property.26 Thiophene is the smallest fluo-
rescent nucleotide, which shows a weak fluorescence property.27
⇑
Corresponding author at: Department of Chemistry, Research Institute of
Physics and Chemistry, Chonbuk National University, Jeonju 561-756, South Korea.
E-mail address: yseo@jbnu.ac.kr (Y.J. Seo).
https://doi.org/10.1016/j.bmcl.2018.04.058
0960-894X/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Le B.H., Seo Y.J. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.058

2
B.H. Le, Y.J. Seo / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Scheme 1. Concept of rolling circle amplification with fluorescent nucleotide.
First, we synthesized fluorescent nucleotide triphosphate based
hybridized with miRNA 24-3P and could form a circular DNA with
ligation using T4 DNA Ligase. To confirm the circular DNA forma-
tion, we used EXO 1 enzyme, which could cleave single-stranded
DNA selectively but could not cleave the circular DNA. Figure 1a
shows clear evidence of RCT 1 circular DNA formation.
To validate the rolling circle amplification using our unnatural
fluorescent nucleotides, we added phi 29 DNA polymerase with
dATP, dGTP, and dCTP including each fluorescent nucleotide
(dUpyrTP, dUanthTP, and dUthioTP) into the RCT 1 circular
DNA, which is complementary to the miRNA24-3P sequence. We
examined agarose gel electrophoresis to confirm the RCA product
by using enzymatic synthesis (Fig. 1b). Interestingly, it showed a
size-dependent incorporation and extension pattern. From
dUpyrTP and dUanthTP, we did not observe any amplified DNA
product, while we could observe amplified DNA product using
dUthioTP. Only by using dUthioTP did we observe over 10 kb
RCA DNA product. From this result, we noted that only the
on the Yoshiuki method28 via several steps and confirmed the syn-
thesis using NMR and mass spectrometry (see Supporting Informa-
tion). Then, we examined the enzymatic incorporation properties
of these fluorescent nucleotides using the RCA system. For this,
we designed RCT 1 as a rolling circle template, which is the com-
plementary sequence-targeting miRNA 24-3P (Table 1). RCT 1
Table 1
Oligonucleotide sequences that were used for this study.
Name
Sequences
RCT 1
50-aPho-TGA ACT GAG CCA ACT GCT GCT GCT GCT GCT
GCT GCT GCT GCT GAC TGT TCC TGC
50- ugg cuc agu uca gca gga aca g
miRNA 24-3P
ORN 1
miRNA 21
50- ugg cuc agu ucu gca gga aca g
50- uag cuu auc aga cug aug uug a
a
Pho:Phosphate
Fig. 1. RCA reaction using dUpyrTP, dUanthTP, and dUthioTP. a) Denaturing polyacrylamide gel data to confirm the ligated reaction. Lane 1: miRNA 24-3P; Lane 2: template
RCT 1; Lane 3: miRNA 24-3P + RCT 1 ligated with T4 ligase; Lane 4: miRNA 24-3P + RCT 1 ligated with T4 ligase and treated with Exo I nuclease. b) and c) Agarose gel data,
confirming the success of the RCA reaction (see Supporting Information for detail RCA reaction condition). M: Marker 1 kb; lane 1: miRNA 24-3P + RCT 1 + T4 ligase and RCA
reaction with dNTP; lane 2: RCA reaction with dUpyrTP, dATP, dCTP, dGTP; lane 3: RCA reaction with dUantTP, dATP, dCTP, dGTP; lane 4: RCA reaction with dUthioTP, dATP,
dCTP, dGTP. d) Time dependent fluorescence spectra to determine the effect of dUthioTP. All RCA reactions were prepared with dNTP and including 5 mM dUthio. All samples
were excited at 354 nm.
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B.H. Le, Y.J. Seo / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
a)
b)
400
350
300
250
200
150
100
10-9
10-1
10-3
10-6
miRNA 24-3P concentration (uM)
Fig. 2. Sensitivity data of dUthioTP-RCA system for the detection of miRNA24-3P. a) Agarose gel data confirming the RCA reaction (M: Marker 1 kb; Lane 1: 10ꢀ1; Lane 2:
10ꢀ3; Lane 3:10ꢀ6; Lane 4: 10ꢀ9 mM of target miRNA 24-3P; b) concentration-dependent (10ꢀ9, 10ꢀ6, 10ꢀ3 and 10ꢀ1 mM of target miRNA 24-3P) fluorescence data using
dUthioTP-RCA. All samples were excited at 354 nm. Fluorescence signal change (F-F0), where F (with target miRNA 24-3P) and F0 (without target miRNA 24-3P) are the
fluorescence intensities from the RCA product.
small-sized fluorescent nucleotide could be recognized by the phi
29 DNA polymerase and may be used for the direct labeling of
the RCA reaction.
For the direct labeling, we examined the fluorescence properties
of this dUthioTP-RCA system (Fig. 1c). Interestingly, it showed a
fluorescence signal corresponding to DNA amplification product.
Interestingly, we also observed the DNA gel formation during the
RCA reaction. Thus, we checked if this fluorescence signal is from
the incorporation and extension of our dUthioTP during the rolling
circle amplification or just from the simple interaction of dUthioTP
monomer with the DNA gel. To demonstrate this, we simply added
dUthio nucleoside monomer with dNTP during the rolling circle
amplification. However, we could not observe any increased fluo-
rescence (Fig. 1d). From this experiment, we confirmed that the
fluorescence is from the incorporation and extension of dUthioTP
into the RCA DNA.
Next, to evaluate the sensitivity of this dUthioTP-RCA probing
system, we measured the fluorescence spectra depending on
Fig. 3. Selectivity data of dUthioTP-RCA for the detection of miRNA 24-3P. a) Denaturing polyacrylamide gel data confirming the circular DNA structure of RCT 1 (lane 1:
miRNA 24-3P, lane 2: RCT 1; lane 3: miRNA 24-3P + RCT 1 + T4 ligase; lane 4: miRNA 24-3P + RCT 1 + T4 ligase + Exo I nuclease; lane 5: ORN 1; lane 6: ORN 1 + RCT 1 + T4
ligase; lane 7: ORN 1 + RCT 1 + T4 ligase + Exo I nuclease; lane 8: miRNA 21; lane 9: miRNA 21 + RCT 1 + T4 ligase; lane 10: miRNA 21 + RCT 1 + T4 ligase + Exo I nuclease. b)
Agarose gel data, confirming the success of the RCA reaction (M: Marker 1 Kb; lane 1: miRNA 24-3P + RCT 1 + T4 ligase + phi 29 DNA polymerase; lane 2: ORN 1 + RCT 1 + T4
ligase + phi 29 DNA polymerase; lane 3: miRNA 21 + RCT 1 + T4 ligase + phi 29 DNA polymerase). c) Fluorescence spectra of dUthioTP-RCA system alone and in the presence
of the target miRNA 24-3P, mismatched miRNA (ORN 1) and random target (miRNA 21). All samples were excited at 354 nm.
Please cite this article in press as: Le B.H., Seo Y.J. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.058

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B.H. Le, Y.J. Seo / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
different amounts of miRNA24-3P from uM to aM. To measure
this, we checked whether the RCA product depends on the concen-
tration of miRNA 24-3P (Fig. 2a). Figure 2a shows that the amount
of RCA product depends on the amount of miRNA24-3P. We also
measured that the fluorescence spectra, with the expectation that
the dUthioTP incorporation induces different fluorescence signals
depending on the concentration of miRNA24-3P (Fig. 2b). Figure
2b exhibited a clear increased fluorescence signal even using a
low concentration of miRNA24-3P. From this result we confirm
that our direct incorporation of dUthioTP with the RCA probing
system could discriminate the signal without and with the target
miRNA 24-3P even at low concentrations.
Finally, to assess specificity, we designed and synthesized ORN
1 and miRNA21 as a one base mismatch and other miRNA
sequences. Figure 3a shows the selective circular DNA formation.
We observed circular DNA formation with miRNA24-3P, while
we could not observe circular DNA formation with ORN 1 and
miRNA 21. We also observed RCA product with miRNA24-3P,
but we could not observe any RCA product with ORN 1 and
miRNA21 sequences, which shows clear evidence of the selective
detection of target miRNA24-3P. Finally, we measured the fluores-
cence spectra to confirm the selectivity, and we could not observe
any fluorescence signal increase with ORN 1 or miRNA21, while
we could observe a dramatic fluorescence increase with target
miRNA24-3P. From all of these results, we believe that our direct
labeling system using dUthioTP-RCA could be used for the detec-
tion miRNA24-3P with simple processing and low cost.
In conclusion, to achieve a simple process for the detection of
miRNA 24-3P, we tried to develop a fluorescent nucleotide that
is recognized by phi29 DNA polymerase. For this purpose, we
designed and attached several different sizes of fluorescent sub-
strate at the 50 position of deoxy uridine bases such as thiophene
(dUthioTP), anthracene (dUanthTP), and pyrene (dUpyrTP). We
screened the incorporation and extension capability of these fluo-
rescent nucleotides during rolling circle amplification using phi 29
DNA polymerase. Among these, only dUthioTP exhibited a high
incorporation and extension capability into the rolling circle DNA
product. This dUthioTP-RCA system could be used to detect the
miRNA 24-3P, which is related with PRRSV. We also examined
the sensitivity and selectivity using this dUthioTP-RCA probing
system and confirmed that this system could detect miRNA 24-
3P with high sensitivity. We also observed that this dUthioTP
probing system could discriminate the miRNA 24-3P from one
base (ORN 1) and random miRNA 21. Thus, we believe that
dUthioTP-RCA probing system is very efficient for the detection
of miRNA 24-3P with high sensitivity and selectivity, and does
not require any other extra labeling step, which reduce a compli-
cated time-consuming process and high cost.
Acknowledgments
We thank the NRF project of Korea (2017R1A2B4002398) for
financial support.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.04.058.
References
1. Ambros V. Cell. 2001;107:823.
2. Bartel DP. Cell. 2004;116:281.
3. Harfe BD. Curr Opin Genet Dev. 2005;15:410.
4. Esquela-Kerscher A, Slack FJ. Nat Rev Cancer. 2006;6:259.
5. Tricoli JV, Jacobson JW. Cancer Res. 2007;67:4553.
6. Zhang Y, Yang Q, Wang S. Cancer Chemother Pharmacol. 2014;74:1105.
7. Rabinowits G et al. Clin Lung Cancer. 2009;10:42.
8. Lunney JK, Fang Y, Ladinig A, et al. Annu Rev Anim Biosci. 2016;4:129.
9. Shuqi X, Xue W, Huaibao N, et al. J Virol. 2015;89:4494.
10. Chen C, Ridzon DA, Broomer AJ, et al. Nucleic Acids Res. 2005;33:e179.
11. Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM. RNA.
2005;11:1737.
12. Valoczi A, Hornyik C, Varga N, Burgyan J, Kauppinen S, Havelda Z. Nucleic Acids
Res. 2004;32:175.
13. Quavi AJ, Kindt JT, Gleeson MA, Bailey RC. Anal Chem 2011;83:5949.
14. Qinfeng X, Anping C, Ling FZ, Chun YZ. Anal Chem. 2012;84:10845.
15. Bin CY, Yu QL, Bang CY. J Am Chem Soc. 2012;134:5064.
16. Mohsen MG, Kool ET. Acc Chem Res. 2016;49:2540.
17. Zhou Y, Huang Q, Gao J, Lu J, Shen X, Fan C. Nucleic Acids Res. 2010;38:e156.
18. Taku M, Jun S, Makoto K. Nucleic Acids Res. 2009;37:19.
19. Jonstrup SP, Koch J, Kjems J. RNA. 2006;12:1747.
20. Mestdagh P, Feys T, Bernard N, et al. Nucleic Acids Res. 2008;143:32.
21. Wang L, Luo YZ, Zhang L, Jiao XM, Wang MB, Fan YL. Nucleic Acids Res.
2008;36:149.
22. Li J, Deng T, Chu X, et al. Anal Chem. 2010;82:2811.
23. Hong C, Baek A, Hah SS, Jung W, Kim DE. Anal Chem. 2016;88:2999.
24. Le BH, Koo JC, Joo HN, Seo YJ. Bioorganic Med Chem. 2017;25:3591.
25. Hurley DJ, Tor Y. J Am Chem Soc. 2002;124:3749;
Mahara A, Iwase R, Sakamoto T, Yamana K, Yamaoka T, Murakam A. Angew
Chem Int Ed. 2002;41:3648;
Kim IS, Seo YJ. Bioorg Med Chem Lett. 2014;24:1589;
Park JW, Seo YJ, Kim BH. Chem Commun. 2014;50:52;
Kim IS, Seo YJ. Tetrahedron Lett. 2014;55:1461.
26. Guntas G, Hallett RA, Zimmerman SP, et al. Proc Natl Acad Sci USA.
2015;112:112.
27. Robin MP, O’Reilly RK. Chem Sci. 2014;5:2717;
Noé MS, Sinkeldam RW, Tor Y. J Org Chem. 2013;78:8123.
28. Hollenstein M. Molecules. 2012;17:13569.
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