A highly sensitive nucleic acid stain based on amino-modified tetraphenylethene: The influence of configuration

Article abstract of DOI:10.1039/c4cc02671c

We designed and synthesized a new amino-functionalized tetraphenylethene (TPE) derivative as a highly sensitive dye for the detection of dsDNA and oligonucleotide in both solution and a gel matrix. We further revealed that the cis configuration dye showed

Full text of DOI:10.1039/c4cc02671c

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A highly sensitive nucleic acid stain based
on amino-modified tetraphenylethene:
the influence of configuration†
Cite this: Chem. Commun., 2014,
50, 6494
Received 11th April 2014,
Accepted 1st May 2014
Li Xu, Zece Zhu, Xiang Zhou, Jingui Qin and Chuluo Yang*
DOI: 10.1039/c4cc02671c
www.rsc.org/chemcomm
We designed and synthesized a new amino-functionalized tetra- cost-effective and highly sensitive dyes for the quantitation and
phenylethene (TPE) derivative as a highly sensitive dye for the detection of nucleic acids in a gel matrix.
detection of dsDNA and oligonucleotide in both solution and a gel
In recent years, tetraphenylethene (TPE) derivatives have
matrix. We further revealed that the cis configuration dye showed a been widely studied in the field of cell labeling,9 DNA and
much higher sensitivity than its trans isomer for the first time.
protein detection.10 Unlike the conventional self-quenched
fluorescent probes, TPE-based dyes are non-emissive as
Gel electrophoresis is one of the most valuable techniques for unassociated monomers, but become strongly fluorescent upon
the separation of nucleic acids in life sciences.1 The visualiza- aggregation at high concentrations.11 This novel aggregation-
tion of nucleic acid bands in a gel matrix relies on the genera- induced emission (AIE) effect is proposed to be caused by
tion of a contrast image through a readout species, which restriction of intramolecular rotation (RIR) that prohibits
includes organic fluorescent dyes, silver stain, zinc-imidazole energy dissipation via non-radiative channels, leading to high
etc.2 In particular, fluorescent probes are usually preferred in quantum yields in aggregated states.12 Tang et al. felicitously
view of sensitivity and convenience. Commercial dyes widely designed a series of TPE derivatives with tetraalkylammonium
used as markers in gel electrophoresis are based on fluorescent cations as ‘‘light up’’ probes for DNA detection.10d These probes
enhancement triggered by intercalating into or groove binding bind to DNA through electrostatic interaction, quite unlike the
to DNA, such as Ethidium Bromide (EB), Gel Star, and SYBR classical intercalators or groove binders, and thus they were
stains.2b,g Among these fluorescent dyes, EB is a commonly further used as a new kind of DNA stain for gel electrophoresis
used nucleic acid stain because of its high sensitivity and low with the lowest detection limit of 0.25 mg.7a In addition, it is
cost.3 But EB is reported to be a toxic and mutagenic agent also reported that some TPE-based probes are cytocompatible
because of its intercalating property upon binding to nucleic and pose no threat of toxicity to living cells.10d,e
acids.4 To be specific, EB has been shown to inhibit replication
In this communication, aiming to develop simple, universal
in several organisms by interfering with DNA synthesis5 and and highly sensitive dyes for the detection of nucleic acids in
causes frame shift mutations in bacteria.4d,6 Besides, due to its a gel matrix, we designed and synthesized a new amino-
binding mode of intercalating into DNA, EB is less sensitive in functionalized TPE derivative (Scheme 1). We expected that
detecting single-stranded DNA, especially oligonucleotides the introduction of an amino group would strengthen the
without secondary structures.7 Some alternatives of EB, such interaction between the dye and nucleic acid by the formation
as SYBR-based dyes, have been found to be highly sensitivite of a hydrogen bond, and thus the new dye could detect not only
but less carcinogenic.8 However, the availability of these pro- dsDNA but also oligonucleotides with high sensitivity in both
ducts at high price is disadvantageous to high-throughput solution and a gel matrix. We further revealed that the cis
detection. Thus, it is a pressing concern to develop less-toxic, configuration dye showed a much higher sensitivity than its
Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials,
Hubei Key Lab on Organic and Polymeric Optoelectronic Materials, Department of
Chemistry, Wuhan University, Wuhan, 430072, People’s Republic of China.
E-mail: clyang@whu.edu.cn
† Electronic supplementary information (ESI) available: Experimental details,
including experimental procedures, structural characterization data, fluorescence
spectra. CCDC 992549. For ESI and crystallographic data in CIF or other electro-
Scheme 1 The structures of Z-N2TPE and E-N2TPE.
nic format see DOI: 10.1039/c4cc02671c
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trans isomer for the first time. The cis configuration dye can
stain 10 ng of short oligonucleotide with only 20 nt, and 1 ng of
dsDNA with 300 bp in polyacrylamide gel electrophoresis
(PAGE). The detection limits are significantly lower than those
of the commonly used nucleic acid stain of EB.
The synthetic route of N2TPE is shown in Scheme S1 (ESI†).
Intermediate 1 was synthesized by substitution of 4-hydroxy-
benzophenone with 1,2-dibromoethane. Subsequently, inter-
mediate 2 was obtained through titanium-catalyzed McMurry
coupling reaction. The azide compound 3 obtained by the
reaction of 2 with NaN3 was reduced to give the final product,
named N2TPE, which is obviously a mixture of the cis and trans
isomers. The pure Z-N2TPE and E-N2TPE were separated by
careful chromatography with yields of 41% and 44%, respec-
tively. The pure isomers showed doublets at ca. 6.6 (H1) and
6.9 (H2) ppm, as well as triplets at ca. 3.9 (H6) and 3.0 (H7) ppm
assigned to the ethylene group, while their mixture showed
mutiplets due to the overlap of these peaks (Fig. S1, ESI†). The
configuration of the cis isomer (Z-N2TPE) was further deter-
mined by single-crystal X-ray analysis of its dihydrochloride
(Fig. S1, ESI†).
Fig. 2 Fluorescence titration of X30 to Z-N2TPE (A) and E-N2TPE (B) in
deionized water. Plot of I/I0 À 1 at 480 nm versus the oligonucleotide
(X10, X20, and X30) concentration of Z-N2TPE (C) and E-N2TPE (D). I0
=
emission intensity in the absence of oligonucleotides. [Z-N2TPE] = 10 mM.
[E-N2TPE] = 10 mM; lex = 330 nm, lem = 480 nm, error bars are ÆSD. X10,
X20, X30 are synthetic oligonucleotides with a length of 10 nt, 20 nt and
30 nt, respectively.
N2TPE was initially exploited to detect DNA. Fig. 1A presents
the fluorescence response of N2TPE to X30 (synthetic oligo- oligonucleotide at the same concentration, the fluorescent
nucleotide with 30 nt). Addition of X30 in the concentration intensity of Z-N2TPE was about two times higher than that of
range from 0 to 0.5 mM led to a progressive increase in the E-N2TPE. When the X30 concentration reached 0.5 mM, the
emission intensity of N2TPE at 480 nm. The FL intensity is fluorescent enhancement of Z-N2TPE was 143 fold its original
61 fold its original value when the DNA concentration is 0.5 mM value, in contrast the fluorescent enhancement of E-N2TPE was
(Fig. 1B). The non-emissive N2TPE became highly luminescent only 8 fold. This should be attributed to neighbouring group
upon binding to DNA. We speculated that the intramolecular participation. The cis configuration with two close interaction
motions of the N2TPE were restricted through both hydrogen- centers could bind DNA with higher affinity than its trans
bond and electrostatic interaction between the N2TPE and isomer. To study the effect of DNA chain length, oligonucleo-
DNA, consequently the fluorescence became ‘‘turn on’’.
tides with different lengths were tested. As shown in Fig. 2C
Similar to other TPE derivatives used in DNA detection,10 and D, with the reduction of the length of the oligonucleotides
N2TPE is a mixture of cis and trans configurational isomers. from X30 to X10, the fluorescent enhancement was substan-
The stereochemistry was usually ignored in the study of tiallly decreased. When the oligonucleotide length is as short as
TPE-based compounds due to the difficulty of separation after X10 with only 10 nt, it affords less interaction sites with
preparation by the McMurry reaction. Recently, Tang et al. Z-N2TPE or E-N2TPE, and thus the aggregation may not be
reported pure cis/trans TPE derivatives, but their difference as enough to turn on the fluorescence. We also consider ctDNA
bio/chemosensor is still to be explored.13 By the aid of the pure (calf thymus DNA, a natural dsDNA) as a model analyte for
Z-N2TPE and E-N2TPE isomers, we explored the configura- dsDNA detection (Fig. S2–S3, ESI†).
tional effects on DNA detection. The fluorescence spectra were
The ‘‘turn on’’ fluorescent response and high sensitivity of
recorded in deionized water (Fig. 2A and B). Z-N2TPE is almost the newly designed TPE derivatives in detecting DNA in aqu-
not emissive, while E-N2TPE is weak emissive. Upon addition of eous solution inspired us to further explore their application as
a DNA stain in gel electrophoresis. We still used the pure
Z-N2TPE and E-N2TPE as the stain to investigate the config-
urational effect (Fig. 3A and B). For comparison, we also tested
the widely used stain of EB (Fig. 3C) and the mixture of Z/E
isomers under the identical conditions (Fig. 3D). After running
polyacrylamide gel electrophoresis (PAGE) in a tris-boric acid-
EDTA (TBE) solution, the gel was stained by 10 mM solution.
We firstly tested the ssDNA as shown in lane 1–3 of Fig. 3A–
D. After staining by Z-N2TPE, the bands of X20 and X30 with
10 ng loading can be seen, while the band of X10 is not
detectable. With the increasing amount of DNA from 10 ng to
40 ng, the bands of X20 and X30 become distinct to identify,
while 40 ng of X10 is still undetectable (Fig. 3A, lane 1 to 3).
Fig. 1 (A) Fluorescence titration of X30 to N2TPE in deionized water.
(B) Plot of I/I0 À 1 at 480 nm versus X30 concentration. I0 = emission intensity
in the absence of oligonucleotide. X30 is a synthetic oligonucleotide with
30 nt. [N2TPE] = 10 mM; lex = 330 nm, lem = 480 nm, error bars are ÆSD.
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are collected in Table 1, in which the results of EB accord with
those reported in the literature.7 In the range of 10–50 bp
dsDNA, the detection limit of Z-N2TPE is ca. one-third the
magnitude of EB. The lowest detectable limits are 1 ng and 4 ng
in the range from 75 to 300 bp for Z-N2TPE and EB, respec-
tively. It is obvious that Z-N2TPE is superior to the commonly-
used commercial DNA stain of EB. We finally used the mixture
of Z-N2TPE and E-N2TPE (1 : 1 ratio by weight) as the stain, and
observed an average result between Z-N2TPE and E-N2TPE
(Fig. 3D). This result means that the mixture of Z/E isomers
could also be used as the DNA stain. Though the sensitivity
is not as high as the case of Z-N2TPE, there is no need of
separation between the Z/E isomers which would be advanta-
geous to its real application in terms of the stain cost.
In conclusion, we developed a new amino-functionalized
TPE derivative as a simple, universal and highly sensitive dye
for the detection of nucleic acids in a gel matrix. By the aid of
Fig. 3 Fluorescence staining of nucleic acids in polyacrylamide gels by
Z-N2TPE (A), E-N2TPE (B), EB (C), and N2TPE (D). Oligonucleotide size
markers (X10, X20, and X30) with equal nano-gram amounts of each
oligounucleotide were loaded in lane 1 to 3. Lane 1: 10 ng, lane 2: 20 the pure cis and trans configuration TPE isomers, we demon-
ng, lane 3: 40 ng per band; ultra low range dsDNA ladder (10, 15, 20, 25, 35,
strated the significant differences of Z/E isomers in DNA
detection and as nucleic acid stains for the first time. The cis
configuration dye showed a much higher sensitivity than its
50, 75, 100, 150, 200, and 300 bases) were loaded in lane 4 to 8. Lane
4: 1 ng, lane 5: 2 ng, lane 6: 4 ng, lane 7: 6 ng, lane 8: 12 ng per band at
300 bp. Concentration of dyes: 10 mM. Staining time: 30 min.
trans isomer. This reveals that many other stereo isomers may
differ in affinity to bind many analytes. The ultra-low detection
In contrast, the bands of X20 and X30 stained by E-N2TPE or limits and universality superior to EB make it promising in real
the reference EB are not clear to identify even with a loading application.
amount of up to 40 ng (Fig. 3B and C, lane 3). The superior
We are grateful for financial support from the National
sensitivity of Z-N2TPE over E-N2TPE for the DNA stain in the Science Fund for Distinguished Young Scholars of China
gel accords with the results of DNA detection in an aqueous (No. 51125013), the program for Changjiang Scholars and Inno-
solution (Fig. 2C and D).
vative Research Team in University (IRT1030), the Research Fund
We then tested dsDNA as shown in lane 4–8 of Fig. 3A–D. for the Doctoral Program of Higher Education of China
Similar to the case of the ssDNA length, with the increasing (No. 20120141110029), and the PhD independent research pro-
dsDNA fragment size from 10 to 300 bp, the stained bands jects of Wuhan University (No. 2012203020214).
become gradually clear. After staining by Z-N2TPE, the band of
50 bp dsDNA can be seen with only 3.5 ng, and becomes
distinct with increasing amount of DNA (Fig. 3A, lane 4 to 8).
By contrast, the band of 50 bp dsDNA stained by E-N2TPE is
still not clear even with a DNA amount of up to 42 ng (Fig. 3B,
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