Propargylamine-selective dual fluorescence turn-on method for post-synthetic labeling of DNA

Article abstract of DOI:10.1039/d0cc00255k

We have developed a propargylamine-selective dual fluorescence turn-on system, using ylidenemalononitrile enamines, for post-synthetic DNA labeling, allowing the direct monitoring of DNA using dual emission in living cells.

Full text of DOI:10.1039/d0cc00255k

View Article Online
View Journal
ChemComm
Chemical Communications
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. J. Seo, V.
Nguyen and A. Pandith, Chem. Commun., 2020, DOI: 10.1039/D0CC00255K.
Volume 54
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Number
January 2018
Pages 1-112
1
4
ChemComm
Chemical Communications
rsc.li/chemcomm
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
S. J. Connon, M. O. Senge et al.
Conformational control of nonplanar free base porphyrins:
towards bifunctional catalysts of tunable basicity
rsc.li/chemcomm

Please do not adjust margins
Page 1 of 5
ChemComm
View Article Online
DOI: 10.1039/D0CC00255K
Journal Name
COMMUNICATION
Propargylamine-selective dual fluorescence turn-on method for
post-synthetic labeling of DNA
Van Thang Nguyena, Anup Pandithb, and Young Jun Seoa,b,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We have developed a propargylamine-selective dual fluorescence using an amino-functionalized DNA. We wanted our compounds to
turn-on system, using ylidenmalononitrile enamines, for post- display no (or very weak) fluorescence prior to labeling, yet strong
synthetic DNA labeling, allowing the direct monitoring of DNA dual fluorescence afterward. This bioorthogonal reaction mediated
using dual emission in living Cell.
fluorescence turn-on method would, thereby, minimize false-positive
signals while simplifying the labeling process and providing a
convenient means of monitoring the fluorescence signal directly.
For this purpose, we employed the reaction between
ylidenemalononitrile enamines and primary amines, as developed
previously by the McQuade group.39,40 Our strategy for developing
nucleoside- and oligonucleotide-based fluorescence turn-on systems
was to use ylidenemalononitrile enamines and amino-presenting
nucleosides or oligonucleotides (Scheme 1). The functionalization of
oligonucleotides can be difficult because these complex structured
macrobiomolecules often hide their reaction sites. Thus, we suspected
that a propargylamino group would be sufficiently long to ensure the
Fluorescence DNA labeling has been applied in many bioimaging
studies,1–3 including investigations of the dynamics of intracellular
DNA4-5 and RNA,6–8 DNA structural transformations,9–13 gene
expression,14-16 and various gene diagnostics.17–18 Although many
methods have been developed, post-synthetic fluorescence labeling
would be the most efficient biocompatible method. Over the several
years numerous post synthetic labeling method have been reported
using inverse electron demand Diels–Alder (iEDDA) reactions,19-20
diazyrin based photo cross linking,21-22 and Suzuki–Miyaura cross
coupling for RNA labeling.23
Ideally, post-synthetic fluorescence DNA labeling would result in
fluorescence properties appearing only when the target DNA is
labeled, and not on prior to its labeling. Furthermore, ideal post-
synthetic fluorescence DNA labeling should be shown dual emission
that provide accurate localization and eliminate the interrupted
background signal in in vivo imaging.
post-reaction
labeling
of
the
oligonucleotides
with
ylidenemalononitrile enamines.
Most commercially available fluorescence labeling methods use
compounds that already exhibit strong single fluorescence prior to
labeling of the DNA32-38 such that some fluorescent residues can
remain after labeling to elicit false-positive signals. In this point of
view, post synthetic fluorescence turn on system would be promising
labeling method.
A lot of fluorescence turn on system on DNA have been developed
using tetrazine-modified dyes24-27 and azide28 and alkyne-modified
dyes based on coumarins,29-30 and naphthalimides.31 However it still
demands more diverse types of fluorescence turn on system with
different functinal group. Propargyl amine directing fluorescence turn
on system would be alternative labeling method instead of tetrazine
and azide based fluorescence turn on system.
In this regard, our goal for this study was to develop a
bioorthogonal reaction mediated dual fluorescence turn-on method
Scheme 1. (A) Structures of 5´-(propargylamino)deoxyuridine (AmdU) and the
ylidenemalononitrile enamines tested in this study. (B) Design and synthesis of
fluorescence nucleosides. (C) Synthesis of functionalized oligonucleotides.
aDepartment of Bioactive Material Sciences, Jeonbuk National University, South
Korea.
bDepartment of Chemistry, Jeonbuk National University, South Korea.
* Fax: +82-63-270-3408; Tel.: +82-63-270-3417; E-mail: yseo@jbnu.ac.kr.
Electronic Supplementary Information (ESI) available: [Full experimental details,
characterization of compounds, UV absorption, circular dichroism spectra, UV
melting curves of oligonucleotides]. See DOI: 10.1039/x0xx00000x
As a potentially suitable amino-derived nucleoside, we synthesized
5´-(propargylamino) deoxyuridine (AmdU). Next, we reacted AmdU
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 5
COMMUNICATION
Journal Name
with three different ylidenemalononitrile enamines (P1–P3) and oligonucleotide sequence (ODN 1) (Table S1). We confirmed the
View Article Online
obtained the three novel deoxyuridine-based nucleosides duP1, duP2, propargylamino oligonucleotide using DOMI: 1A0L.10D3I9-/TDO0CFC00m25a5sKs
and duP3, the structures of which we confirmed using NMR spectrometry (Figure S7).
spectroscopy and high-resolution mass spectrometry ( Schemes S1 -
S5 ).
We chose the P3, which induced promising fluorescence properties
when it forms the nucleoside duP3, for reaction with propargylamino-
When we examined the photophysical properties of the three new presenting oligonucleotide. The optimized reaction conditions
nucleoside derivatives (duP1–duP3), we found, gratifyingly, that involving MeCN and phosphate buffer (1:1) as the solvent system and
they all displayed high fluorescence despite their precursors P1–P3 a ratio between the propargylamino-presenting oligonucleotide and
not exhibiting fluorescence or only weak fluorescence (Figure 1). P3 of 1:10—provided a high yield of the oligonucleotide product
Each of the ylidenemalononitrile enamine–reacted nucleosides duP1– (Oligo P3), which was purified using HPLC.
duP3 exhibited different fluorescence properties: duP1 displayed
fluorescence at λmax=470 nm (blue-green); duP2 at λmax = 525 nm
(green); and duP3, interestingly, exhibited dual emission at λmax= 476
We used radio-labeling method to confirm the success of this
reaction because of its high sensitivity toward even small structural
changes. Indeed, Figure 2A reveals the different mobilities of the
nm (λex = 420nm) and at λmax= 570 nm (λex = 510 nm). Measurements
propargylamino-presenting oligonucleotide and the products Oligo
of the fluorescence quantum yields of these three new fluorescent
P1–P3. We confirmed the structures of these products by measuring
their MALDI-TOF mass spectra (Figures 2B and S8). These data
confirmed that selective reactions had occurred between the
propargylamino group and the ylidenemalononitrile enamines, and
that no such reactions occurred with the other free amino groups of
the oligonucleotides.
nucleoside derivatives revealed that duP3 had a much higher quantum
yield ( = 0.30) than those of duP2 ( = 0.02) and duP1 ( = 0.001)
(Table S2).
A
B
Figure 2. (A) Autoradiogram of the denaturing polyacrylamide gel
electrophoresis of the reactions of 32P-5´-end–labeled ODN1 (containing 1 µM
AmdU) in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM
KH2PO4, pH 7.4) and the three ylidenemalononitrile enamines (0.1 mM in
Figure 1. Fluorescence spectra and photographs (under irradiation at 365 nm) MeCN). These mixtures had been stirred overnight. Lane 1: 32P-5´-end–
of the products of the reactions of AmdU with the ylidenemalononitrile labeled ODN1; lane 2: reaction with P1; lane 3: reaction with P2; lane 4:
enamines (B) P1, (C) P2, and (D) P3 in MeCN, stirred overnight at room reaction with P3; lane 5: 32P-5´-end–labeled ODN1. (B) MALDI-TOF mass
temperature.
spectrum of the reaction product formed between ODN1 and P3; Oligo P3:
calcd m/z 4766.28, found 4767.50.
The fluorescence properties of duP1–duP3 varied depending on
the solvent. Although duP1 did not undergo any significant change in
fluorescence upon changing the solvent from MeCN to water (Figure
S1), the fluorescence of duP2 did change accordingly from blue to
green (Figure S2), while duP3 underwent a dramatic change in color
from blue in water to red in various other solvents (Figure S3). Based
on its high quantum yield with dual emission which is useful to verify
the location of fluorescence in the living cell and high sensitivity
toward the solvent, we suspected that duP3 would be promising
fluorescent nucleoside for incorporation into DNA.
We recorded fluorescence spectra to examine whether the
fluorescence turn-on was also occurring with the modified
oligonucleotides. Interestingly, when the propargylamino-presenting
oligonucleotide was added into solutions of the P1–P3, the
fluorescence signals increased dramatically, with different emission
wavelengths (Figure 3A): Oligo P1 exhibited blue fluorescence at
(λmax) 475 nm; Oligo P2 exhibited green fluorescence at (λmax) 515
nm; and Oligo P3 exhibited fluorescence at (λmax) 475 nm according
to a solvent effect in water. Thus, our selective fluorescence turn-on
system could indeed function as a post-synthetic modification method
for the direct labeling of DNA. To confirm the utility of this
fluorescence turn-on system, we employed denaturing PAGE and
observed the fluorescence signals directly by the naked eye at the
same positions as the oligonucleotides (Figure 3B). The fluorescence
gel image was correlated well with Oligo P3, with its blue
fluorescence originating from the pH of the PAGE buffer. Oligo P3
also exhibited pH-dependent fluorescence, similar to that of duP3
(Figure S9).
Next, we investigated whether this fluorescence turn-on process
operated also with oligonucleotides, with the possibility that the
amino groups of deoxycytosine,
deoxyadenosine, and
deoxyguanosine residues might compete with the propargylamino
group. With this concern in mind, we synthesized a trifluoroacetyl-
protected propargylamino-deoxyuridine phosphoramidite (Scheme
S1) and incorporated it into various oligonucleotides using solid phase
synthesis, then deprotected the propargylamino group (using
ammonium hydroxide) and applied general oligonucleotide synthetic
and purification procedures to obtain free propargylamino-presenting
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 5
ChemComm
Please do not adjust margins
Journal Name
COMMUNICATION
A
B
reactions in living fibroblast sarcoma-HT1080 cancerVcieewllAlritincleesO(nslienee
experimental section in supporting information).41 Initially to identify
DOI: 10.1039/D0CC00255K
the suitable concentration of probe P3 per plate of live cell culture we
have performed concentration dependent studies (Figure S11). Based
on that studies residual amines present in per plate of cells cytosol
were identified and background images were processed with
appropriate calibration (intensity based spectral corrections) using
Image J software.
Accordingly, upon incubation of 5 M solution of P3 in fibroblast
sarcoma HT01080 cell lines, it showed very low emission in green
and red channels (Figure 5a). In contrast upon sequential incubation
of P3 (5 M) with small molecule propargyl amine (10.0 eq.) showed
bright fluorescence emission in green and red channel and supported
the amine units undergo reaction with P3 in intracellular conditions
(Figure 5b). Upon treatment of P3, with transfected propargylamino-
presenting oligonucleotide using Lipofectin (TM DOTAP-DOTMA),
showed bright spots in green and red channels and validated effective
bioorthogonal reactions in cancer cell lines (Figure 5c). Higher
concentration of P3 with specified amount of propargylamino-
presenting oligonucleotide showed substantial fluorescence foci spots
with diffused background fluorescence (Figure 5d). We suspect that
foci spot come from Lipofectin(TM) induced specific location of
oligonucleotide in cytosol, while small molecule propargyl amine
with P3 showed random diffused fluorescence.
Figure 3. (A) Emission spectra of the
products of the reactions of ODN1 (0.1 mM) in PBS buffer (137 mM NaCl, 2.7
mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) with the three
ylidenemalononitrile enamines (1 mM in MeCN). These reactions were stirred
overnight. (B) Denaturing PAGE characterization of the product of the
reaction between 0.1 mM ODN1 (containing AmdU) in PBS buffer (137 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) with 0.5 mM P3
(in
MeCN).
Gels
were
stained
with
EtBr.
We recorded time-dependent fluorescence spectra to confirm that
the turn-on system could be applied directly—using ODN 1 (0.1 mM)
and P3. Figure 4 reveals a rational fluorescence increase depending
on the time. This data clearly demonstrate the suitability of directly
monitoring the fluorescence turn-on when applying the
propargylamino-presenting oligonucleotide.
We confirmed the location of oligonucleotide by using blue-bright
field images (Figure S12).
Figure 4. Time-dependent fluorescence spectra of the reaction between
ODN1 (0.1 mM) and P3 (1 mM) in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10
mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). ). I0 means initial fluorescence and I
means
after
fluorescence
turn
on
at
each
time.
To evaluate the kinetics of the reactions, based on fluorescence
emission intensity, concentration dependent studies were performed
in specified interval of time (Figure S10A-10C). Based on results
obtained value was Km = 15.0M/min. Upon plotting the inverse
reciprocal of reactant concentrations verses time scale, we found
linear relationship with k = 0. 36 mM-1min-1 (linear fitting) clearly
supported that reactions followed second order kinetics (Figure
S10D). Hence we believe that reaction followed second order kinetics
under physiological conditions.
We also examined the reactivity of other amines in the nucleobases
using natural oligonucleotide (random ODN) and T15 that doesn’t
have propargyl amine (Figure S15). We observed P3 reacted with
only propargyl amino presenting oligonucleotide (ODN 1) not for the
random sequence which contain several amines in their nucleobases.
Most of amines in their nucleobases on the oligonucleotide must be
hindered sterically and have low nucleophilicity than propargyl
amine. From this result, we confirmed that the reactivity of propargyl
amino presenting oligonucelotide (ODN 1) with P3 is clearly
discriminated from amines in the nucleobase of natural
oligonucleotide.
Figure 5. Fluorescence confocal laser live cell imaging studies of P3 (5 M)
in fibroblast sarcoma-HT1080 cancer cell lines; a) Cells incubated only with
P3, b) cells incubated with Propargyl amine (10 eq.), 60 min. followed by
sequential addition of P3 (5 M) 60 min., c) Cells were incubated with
propargylamino-presenting oligonucleotide with aid of Lipofectin (TM) (1:5
ratio), about 14 h followed by sequential addition of P3 (3 M) d) Cells were
incubated with propargylamino-presenting oligonucleotide with aid of
Lipofectin(TM) (1:5 ratio), about 14 h followed by sequential addition of
increased amount of P3 (15 M). (validations of the background and diffused
signals from the P3 in cell cytosol); e) Cells were incubated with
propargylamino-presenting oligonucleotide with aid of Lipofectin (TM) (1:5
ratio), about 14 h followed by sequential addition of P3 (3 M) in low cell
density cell culture plate (cell counts approximately 800 to 1200 per plates).
Note: Images were collected at 20 m scale, and incubation temperature was
37o C. Each image was notified with minimum 6 consecutive readings in
various locations of each plate with ~2000 cells. Images numbered from 1-6
represented blue, green, red, blue-green (merge 50:50%), blue-red (merge
50:50%) and blue-green-red channels (merge 33:33:33%) respectively.
Upon inspiring the utilities of propargylamino-presenting
oligonucleotide in synthetic biology we initiated bio-orthogonal
We also examined single cell based molecular imaging upon
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 5
COMMUNICATION
Journal Name
6.
7.
8.
9.
C. Domnick, G. Hagelueken, F. Eggert, O. Schiemann and S. Kath-
careful consideration of concentration effects. Propargylamino-
presenting oligonucleotide were transfected in to the low cell density
of cell culture plates. Accordingly, we found clear reactions between
View Article Online
Schorr, Org. Biomol. Chem., 2019, 17, 1805.
DOI: 10.1039/D0CC00255K
D. He, K.-W. Wong, Z. Dong and H.-W. Li, J. Mater. Chem. B.,
2018, 6, 7773.
P3
and
cytosolic
localized
propargylamino-presenting
T. Ozawa, Y. Natori, M. Sato and Y. Umezawa, Nat. Methods.,
2007, 4, 413.
F. He, Y. Tang, S. Wang, Y. Li and D. Zhu, J. Am. Chem. Soc.,
2005, 127, 12343.
I. S. Kim and Y. J. Seo, Bioorg. Med. Chem. Lett., 2014, 24, 1589.
H. Kuhn, V. V. Demidov, J. M. Coull, M. J. Fiandaca, B. D. Gildea
and M. D. Frank-Kamenetskii, J. Am. Chem. Soc., 2002, 124, 1097.
D. Liu and S. Balasubramanian, Angew. Chem., Int. Ed., 2003, 42,
5734.
Y. J. Seo, H. Rhee, T. Joo and B. H. Kim, J. Am. Chem. Soc., 2007,
129, 5244.
J. DeRisi, L. Penland, M. Bittner, P. Meltzer, M. Ray, Y. Chen, Y.
Su and J. Trent, Nat. Genet, 1996, 14, 457.
D. J. Lockhart and E. A. Winzeler, Nature, 2000, 405, 827.
M. Schena, D. Shalon, R. W. Davis and P. O. Brown, Science,
1995, 270, 467.
X. Chen, B. Zehnbauer, A. Gnirke and P.-Y. Kwok, Proc. Nat.
Acad. Sci. U.S.A, 1997, 94, 10756.
oligonucleotide which were identified in green and red channels
(Figure 5e). Upon careful observation through the single cell showed
highly selective turn on response in green and red dual emissive
channels upon incubating transfected propargylamino-presenting
oligonucleotide with P3.
10.
11.
To clarify the Lipofectamine effect that also have amine, we
measured fluorescence confocal laser live cell imaging using only
Lipofectin(TM) with Probe P3 based on previously reported
Lipofectamine metabolism42 and it did not induce any enough
characteristic fluorescence spot (Figure S13). In contrast upon
increment of concentration of propargylamino presenting
oligoucleotide it showed high number fluorescence spots in cell
cytosol, which validated the successful recognition of
propargylamino-presenting oligonucleotide using P3 (Figure S14).
From this result, we believe that presence of Lipofectin (TM) was
almost negligible and propargylamino-presenting oligonucleotide
selective fluorescence turn-on system with P3 is operating in live
fibroblast sarcoma cell line.
12.
13.
14.
15.
16.
17.
18.
Y. D. Lo, N. M. Hjelm, C. Fidler, I. L. Sargent, M. F. Murphy, P.
F. Chamberlain, P. M. Poon, C. W. Redman and J. S. Wainscoat,
N. Engl. J. Med., 1998, 339, 1734.
In conclusion, we have developed a bioorthogonal reaction
mediated dual fluorescence turn-on method for post-synthetic DNA
labeling using an propargylamino-functionalized DNA with
ylidenmalononitrile enamines. We designed and synthesized the
nucleoside derivatives duP1, duP2, and duP3 from the reactions of
propargylamino-presenting deoxyuridine and three different
ylidenmalononitrile enamines, and observed dramatic fluorescence
turn-on properties. Among them, duP3 [ = 0.30; λmax = 565 nm
19.
20.
21.
22.
23.
D. Ploschik, F. Rönicke, H. Beike, R.Strasser and H‐A.
Wagenknecht, ChemBioChem., 2018, 19, 1949.
H. Li, Z. Sun, W. Wu, X. Wang, M. Zhang, X. Lu, W. Zhong and
D. Dai, Org Lett., 2018, 20, 7186.
Z. Qiu, L. Lu, X. Jian and C. He, J. Am. Chem. Soc., 2008, 130,
14398.
C. X. Song and C. He,. Accounts of Chemical Research., 2011, 44,
709.
M. B. Walunj, A. A.Tanpure and S. G. Srivatsan, Nucleic Acids
Research., 2018, 46, e65.
(reddish)], interestingly, exhibited high dual emission property [ λex
=
420nmand λem = 476nm (green), λex = 510nm and λem = 570 nm (red)] 24.
H. Wu, B. T. Cisneros, C. M. Cole and N. K. Devaraj, J Am Chem
Soc., 2014, 136, 17942.
with highly sensitive fluorescence depends on the solvent and pH.
25.
F. Eggert and S. Kath-Schorr, Chem. Commun., 2016, 52, 7284.
Z. He, Y. Chen, Y. Wang, J. Wang, J. Mo, B. Fu, Z. Du, Y. Wang
and X. Zhou, Chem. Commun., 2016, 52, 8545.
A. M. Pyka, C. Domnick, F. Braun, S. Kath-Schorr, Bioconjugate
Chemistry., 2014, 25, 1438.
R. M. Franzini and E. T. Kool, ChemBioChem., 2008, 9, 2981. .
T. Ishizuka, H. S. Liu, K. Ito and Y. Xu Scientific Reports., 2016,
6, 33217.
M. V. Bharathi, M. Chhabra and P. Paira, Bioorg Med Chem Lett.,
2015, 25, 5737.
S. Banerjee, E. B. Veale, C. M. Phelan, S. A. Murphy, G. M. Tocci, L.
J. Gillespie, D. O. Frimannsson, J. M. Kelly and T. Gunnlaugsson,
Chem. Soc. Rev., 2013, 42, 1601.
J. T. George and S. G. Srivatsan, Methods, 2017, 120, 28.
O. Henegariu, P. Bray-Ward and D. C. Ward, Nat. Biotechnol.,
2000, 18, 345.
B. Huy Le, V. T. Nguyen and Y. J. Seo, Chem. Commun., 2019, 55,
2158.
B. H. Le, T.-V. T. Nguyen, H. N. Joo and Y. J. Seo, Bioorg. Med.
Chem., 2018, 26, 4881.
D. Proudnikov and A. Mirzabekov, Nucleic Acids Res., 1996, 24,
4535.
The site-selective reactions of
a
propargylamino-presenting
26.
27.
oligonucleotide with the ylidenmalononitrile enamines also led to
turn-on fluorescence in DNA, with the reactions optimized to produce
the fluorescence turn- on products Oligo P1–P3 (confirmed using
MALDI-TOF mass spectrometry and autoradiograms with PAGE).
Oligo P3 exhibited strong fluorescence and provided a dramatic time-
dependent fluorescence turn-on pattern. We also tried bioorthogonal
reaction using ODN 1 and P3 in the living cancer cell lines and
demonstrated bioorthogonal reaction mediated dual fluorescence
turn-on system (green and red channel) using propargylamino-
presenting oligonucleotide and P3 in the living fibroblast sarcoma-
HT1080 cancer cell lines. This system appears to be a unique method
for the selective turn-on dual emission of propargylamino-presenting
oligonucleotides, and should be useful for the direct and accurate
monitoring of DNA in living Cell.
28.
29.
30.
31.
32.
33.
34.
35.
36.
Acknowledgment
This study was supported by the Basic Science Research Program
through the National Research Foundation of Korea
(2017R1A2B4002398), funded by the Republic of Korea.
37.
38.
39.
P. Z. Qin and A. M. Pyle, Methods, 1999, 18, 60.
M. Schuelke, Nat. Biotechnol., 2000, 18, 233.
A. R. Longstreet, B. S. Campbell, B. F. Gupton and D. T.
McQuade, Org. Lett., 2013, 15, 5298.
A. R. Longstreet, M. Jo, R. R. Chandler, K. Hanson, N. Zhan, J. J.
Hrudka, H. Mattoussi, M. Shatruk and D. T. McQuade, J.
Am.Chem.Soc., 2014, 136, 15493.
I. Ivancova ́, D. -L. Leone, M. Hocek, Current Opinion in Chemical
Biology, 2019, 52, 136.
F. Cardarelli, L. Digiacomo, C. Marchini, A. Amici, F. Salomone,
G. Fiume, A. Rossetta, E. Gratton, D. Pozzi, G. Caracciolo,
Scientific Reports, 2016, 6, 25879.
Supplementary data
Electronic Supplementary Information (ESI) available online: Full
experimental details; characterization of compounds; UV absorption,
fluorescence and Maldi-TOF spectra of the oligonucleotides.
40.
References and notes
41.
42.
1.
2.
J. Liu, Z. Cao and Y. Lu, Chem. Rev., 2009, 109, 1948.
A. B. Neef, L. Pernot, V. N. Schreier, L. Scapozza and N. W.
Luedtke, Angew. Chem., Int. Ed., 2015, 54, 7911.
B. Oliveira, Z. Guo and G. Bernardes, Chem. Soc. Rev., 2017, 46,
4895.
3.
4.
5.
K. M. Dean and A. E. Palmer, Nat. Chem. Bio., 2014, 10, 512.
L. J. Friedman, J. Chung and J. Gelles, Biophys. J., 2006, 91, 1023.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/D0CC00255K
A table of contents entry
We have developed a propargylamine-selective dual fluorescence turn-on system, using ylidenmalononitrile enamines,
for post-synthetic DNA labeling, allowing the direct and accurate monitoring of DNA using dual emission in live Cell.