Nucleic acid-induced aggregation and pyrene excimer formation

Article abstract of DOI:10.1021/ol901607g

Nucleic acid was found to induce the aggregation of the positively charged pyrene probe (compound 1); as a result, strong pyrene excimer emission was observed. The Intensity of the excimer emission was dependent on the concentration of the pyrene probe and the oligonucleotide length, sequence, and concentration. These results suggest a new strategy for label-free nucleic acid-based biosensing applications.

Full text of DOI:10.1021/ol901607g

ORGANIC
LETTERS
2
009
Vol. 11, No. 19
302-4305
Nucleic Acid-Induced Aggregation and
Pyrene Excimer Formation
4
†
,‡
†,‡
†,‡
†
Ruixing Zhang, Dan Tang, Ping Lu, Xiangyu Yang, Dongli Liao,
†
‡
Yujing Zhang, Mingjun Zhang, Cong Yu,* and Vivian W. W. Yam*
‡
,†
,§
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China, Graduate
School of the Chinese Academy of Sciences, Beijing 100039, P. R. China, College of
Animal Science and Veterinary Medicine, Jilin UniVersity, Changchun 130062, P. R.
China, and Department of Chemistry, The UniVersity of Hong Kong, Hong Kong, P. R.
China
congyu@ciac.jl.cn; wwyam@hku.hk
Received July 14, 2009
ABSTRACT
Nucleic acid was found to induce the aggregation of the positively charged pyrene probe (compound 1); as a result, strong pyrene excimer
emission was observed. The intensity of the excimer emission was dependent on the concentration of the pyrene probe and the oligonucleotide
length, sequence, and concentration. These results suggest a new strategy for label-free nucleic acid-based biosensing applications.
Sensing nucleic acids and the analysis of their sequence,
structure, and interactions with regulating factors play a
critical role in basic biological/biomedical research. The
knowledge imparted by these studies has had a profound
influence on diverse areas such as disease diagnostics and
the nucleic acid with a fluorescent probe is needed either
for fluorescence resonance energy transfer (FRET) or
fluorescence quenching studies. Herein we describe the use
of aggregation-induced pyrene excimer fluorescence for
label-free nucleic acid sensing studies.
It is well-known that a number of the planar, aromatic
compounds such as pyrene can form excited dimeric
structures known as excimers. As a result of excimer
formation, a significantly red-shifted, very broad, and almost
1
development of new drugs. In recent years, various new
nucleic acid sensing methods have been developed concomi-
tant with the emergence of novel advanced materials and
2
-10
concepts.
However, in many cases, covalent labeling of
†
State Key Laboratory of Electroanalytical Chemistry, Changchun
Institute of Applied Chemistry, Chinese Academy of Sciences, and Graduate
School of the Chinese Academy of Sciences.
(1) (a) Bloomfield, V. A., Crothers, D. M., Tinoco, I., Jr. Nucleic Acids:
Structures, Properties, and Functions; University Science: Sausalito, 2000.
(b) Nucleic Acids in Chemistry and Biology; Blackburn, G. M., Gait, M. J.,
Eds.; Oxford University Press, Oxford, 1996. (c) The Nucleic Acid Protocols
Handbook; Rapley, R., Ed. Humana Press: Totowa 2000.
‡
College of Animal Science and Veterinary Medicine, Jilin University.
Department of Chemistry, The University of Hong Kong.
§
10.1021/ol901607g CCC: $40.75
Published on Web 09/01/2009
 2009 American Chemical Society

11
structureless emission band can generally be observed. The
use of excimer emission for various sensing applications is
well documented, especially for studies of the physicochem-
1
1c
ical properties of synthetic polymers.
Pyrene excimer
formation has also been used in a number of cases for nucleic
acid related applications, for example, selective sensing of
1
2
potassium or construction of novel nanomaterials. How-
ever, all these methods require that pyrene be covalently
attached to the nucleic acid, which is synthetically quite
demanding. As a result, such methods are time-consuming,
laborious, and very costly.
Figure 1. Structure of compound 1.
Supporting Information). As the concentration of compound
was increased, the I /I value became larger until the point
at which the I /I value reached the maximum. A further
increase of compound 1 concentration caused a decrease of
the I /I value. Figure S2 (Supporting Information) shows
the change in I /I value versus the change in concentration
of compound 1 for poly(dG)25 at pH 7.5 and for poly(dC)25
at pH 5.0 and 8.5. It was found that the maximum I /I value
for poly(dG)25 was much larger than that for poly(dC)25. For
poly(dC)25, the I /I value for pH at 8.5 was considerably
larger than that at pH 5.0.
We have synthesized a positively charged pyrene probe
1
E M
(
compound 1) (Figure 1). The probe shows considerable
E
M
water solubility (>1 mM). When compound 1 was mixed
with the oligonucleotides in an aqueous buffer solution,
remarkable changes in the fluorescence and UV-vis absorp-
tion spectra were observed (Figure 2). For all the oligo-
nucleotides tested, the spectra showed a significant decrease
in emission from the monomeric form of the pyrene probe
and the concomitant appearance of a red-shifted, broad
pyrene emission band with a peak maximum at around 485
nm, which was assigned to pyrene probe excimer emission
E
M
E
M
E
M
E
M
1
1
according to numerous literature reports.
The relative intensity of the pyrene excimer emission
varied significantly as a function of the amount of compound
1
that was added. Our results show that for a fixed poly(dA)25
and poly(dT)25 oligonucleotide concentration of 7.2 µM, the
intensity ratio of pyrene probe excimer emission (485 nm)
to monomer emission (377 nm) (the I
considerably as the concentration of compound 1 was varied.
The trends in the I /I values were quite similar (Figure S1,
E M
/I value) changed
E
M
(
2) (a) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. ReV. 2007,
1
07, 1339–1386. (b) Ho, H.-A.; Najari, A.; Leclerc, M. Acc. Chem. Res.
2
008, 41, 168–178. (c) Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones,
R. M.; Kumaraswamy, S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch,
D.; Mukundan, H.; Rininsland, F.; Shi, X.; Xia, W.; Whitten, D. G. J. Mater.
Chem. 2005, 15, 2648–2656.
(
3) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 10954–10957.
4) Tang, Y.; Feng, F.; He, F.; Wang, S.; Li, Y.; Zhu, D. B. J. Am.
Chem. Soc. 2006, 128, 14972–14976.
5) Park, S.-J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503–
506.
6) Zhang, J.; Song, S.; Zhang, L.; Wang, L.; Wu, H.; Pan, D.; Fan, C.
J. Am. Chem. Soc. 2006, 128, 8575–8580.
7) Li, H.; Rothberg, L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14036–
Figure 2. UV-vis absorption and fluorescence emission spectra
(
of 180 µM of compound 1 alone and following its binding to 7.2
µM of simple oligonucleotide repeats in an aqueous buffer solution
(
(
MOPS, pH 7.5).
1
(
(
1
4039.
E M
As Figure 2 shows, the I /I value varied significantly with
(
8) Tan, W.; Wang, K.; Drake, T. J. Curr. Opin. Chem. Biol. 2004, 8,
the sequence of the nucleic acid added. Poly(dG)25 gave by
far the largest I /I value (12.88). Poly(dA)25 and poly(dT)25
also showed large I /I values (6.20 and 3.03, respectively),
was considerably smaller for poly(dC)25
5
47–553.
E
M
(9) Wang, M.; Zhang, D.; Zhang, G.; Tang, Y.; Wang, S.; Zhu, D. Anal.
Chem. 2008, 80, 6443–6448.
E
M
(
10) (a) Yu, C.; Wong, K. M.-C.; Chan, K. H.-Y.; Yam, V. W.-W.
but the value of I
E M
/I
Angew. Chem., Int. Ed. 2005, 44, 791–794. (b) Yu, C.; Chan, K. H.-Y.;
Wong, K. M.-C.; Yam, V. W.-W. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
(1.46).
1
9652–19657. (c) Yu, C.; Chan, K. H.-Y.; Wong, K. M.-C.; Yam, V. W.-
E M
The I /I value also varied significantly with the concen-
tration of the nucleic acid added. For example, when 3.6,
7.2, or 10.8 µM of poly(dA)25 was mixed with compound 1
W. Chem.sEur. J. 2008, 14, 4577–4584. (d) Yu, C.; Yam, V. W.-W. Chem.
Commun. 2009, 1347–1349. (e) Yu, C.; Chan, K. H.-Y.; Wong, K. M.-C.;
Yam, V. W.-W. Chem. Commun. 2009, 3756–3758.
(
11) (a) Birks, J. B. Rep. Prog. Phys. 1975, 38, 903–974. (b) De
(
Figure S3, Supporting Information), all three curves showed
the same trend as the probe concentration was increased.
The I /I value initially increased, reached the maximum,
Schryver, F. C.; Collart, P.; Vandendriessche, J.; Goedeweeck, R.; Swinnen,
A.; Van Der Auweraer, M. Acc. Chem. Res. 1987, 20, 159–66. (c) Winnik,
F. M. Chem. ReV. 1993, 93, 587–614.
E
M
(
12) (a) Langenegger, S. M.; H a¨ ner, R. Chem. Commun. 2004, 2792–
and then began to decrease. With the increase of the
oligonucleotide concentration from 3.6 to 7.2 and 10.8 µM,
the concentration of compound 1 needed to reach the
2
8
793. (b) Okamoto, A.; Ichiba, T.; Saito, I. J. Am. Chem. Soc. 2004, 126,
364–8365. (c) Nagatoishi, S.; Nojima, T.; Juskowiak, B.; Takenaka, S.
Angew. Chem., Int. Ed. 2005, 44, 5067–5070. (d) Nakamura, M.; Ohtoshi,
Y.; Yamana, K. Chem. Commun. 2005, 5163–5165. (e) Malinovskii, V. L.;
Samain, F.; H a¨ ner, R. Angew. Chem., Int. Ed. 2007, 46, 4464–4467.
maximum I /I
E M
value increased from 150 to 180 and 270
Org. Lett., Vol. 11, No. 19, 2009
4303

µM, respectively. At the same time, the maximum I
E
/I
M
value
The intensity changes of the probe excimer emission upon
mixing with the oligonucleotides were dependent on the
concentration of the probe, as well as the type, concentration,
and length of the oligonucleotides. At a fixed concentration
of the oligonucleotide, an increase of the probe concentration
increased the extent of probe binding to the nucleic acid and
consequently increased the degree of probe aggregation. As
increased from 2.60 to 6.14 and 8.67, respectively. In
addition, a similar trend was observed with the concentration
of compound 1 fixed and the concentration of poly(dA)25
varied (Figure S4, Supporting Information). With increasing
E M
poly(dA)25 concentration, the I /I value increased until it
reached the maximum, after which a further increase of the
oligonucleotide concentration caused a decrease of the I /I
E M
value.
The I /I value also depended on the chain length of the
E M
oligonucleotide (Figure 3). For example, for poly(dA),
reduction of the oligonucleotide length from 25 bases to 20,
E M
a result, the I /I value increased. However, since the
concentration of the nucleic acid was kept constant in the
buffer solution, the total number of negative charges for
probe binding in the assay system did not increase. The
negative charges on the phosphate backbone of the nucleic
acid were largely neutralized up to a certain point. A further
increase of the probe concentration would not be expected
to result in additional binding (that is, the probe reached a
1
5, 10, and 5 bases caused a gradual decrease of the I
E M
/I
value. With only five bases, poly(dA) could not induce the
5
aggregation of the probe, and hardly any excimer emission
was observed.
E M
saturation concentration). Consequently, a decreased I /I
value was observed (Figures S1-S3, Supporting Informa-
tion).
The intensity of the induced excimer emission was
dependent on the type of oligonucleotide used. This effect
is most dramatically demonstrated in Figures 2 and S2
(Supporting Information). It is well-known that poly(dG) can
form G-quadruplex structures as a result of the guanine base
hydrogen bonding and π-π stacking interactions among the
1
four strands of poly(dG). It is also well-known that poly(dC)
can form i-motif structures (Figure S6, Supporting Informa-
1
tion) under an acidic condition. Poly(dG)25 could form
extended four-stranded structures, and the steep rise of charge
density upon G-quadruplex formation would greatly enhance
the aggregation of the probe molecule, and therefore,
E M
Figure 3. Percentage decrease of the I /I value when 180 µM
compound 1 was mixed with poly(dA) of different chain lengths.
The nucleic acid base concentration (dA) was kept constant at 180
µM.
E M
considerably larger I /I values were observed. In contrast,
half of the cytosine bases must be protonated for i-motif
formation. As a result, the total number of negative charges
on the nucleic acid decreases by half, which resulted in
A 25-base oligonucleotide poly(dM)25 (5′-GAT CTG ACG
GTT CAC TAA ACG AGC T-3′) was randomly selected.
Poly(dM)25 and the corresponding duplex DNA were tested
for induced excimer emission (Figure S5, Supporting Infor-
mation). It was found that both poly(dM)25 and the duplex
DNA showed trends similar to the other simple oligonucle-
E M
decreased I /I values.
The intensity of the induced excimer emission was
dependent on the concentration of the oligonucleotide
(Figures S3 and S4, Supporting Information). A higher
concentration of the oligonucleotide resulted in an increase
in the number of electrostatic binding sites available for the
probe molecule, so a higher I /I value is expected. However,
E M
at a fixed probe concentration, if the concentration of the
oligonucleotide exceeded a certain threshold value, a further
increase of its concentration resulted in a decrease of the
E M
otides. For duplex DNA, however, higher I /I values were
generally observed.
Since nucleic acid is a polyanion, the results suggest that
when nucleic acid was mixed with compound 1, electrostatic
attractive force between the positively charged trimethylam-
monium functional group in 1 and the negatively charged
phosphate backbone in the oligonucleotide induced aggrega-
tion of compound 1 in the vicinity of the nucleic acid. That
is, the local concentration of compound 1 around the nucleic
acid increased. Equally important, the positive charge of
compound 1 was largely balanced out by the negative charge
of the nucleic acid. As a result, hydrophobic π-π stacking
interactions between the planar aromatic rings of the probe
molecule dominated. As shown in Figure 2, UV-vis spectral
changes of compound 1 upon mixing with the nucleic acid
clearly indicate ground state aggregation of the probe
molecule, which suggests that the observed excimer is largely
E M
I /I value. The reason is that if the concentration of the
oligonucleotide is too high, the average number of probe
molecules binding to each oligonucleotide decreases. This
effectively “diluted” the number of probes binding to each
oligonucleotide, and a decreased I
Figure S4, Supporting Information).
The induced excimer emission was dependent on the chain
E M
/I value was observed
(
length of the oligonucleotide. A longer oligonucleotide would
carry more negative charges and better induce probe ag-
gregation. Therefore, a decrease of the I /I value was
E M
observed with the decrease in chain length of poly(dA)
(Figure 3). For duplex DNA, although the total number of
negative charges in the system was not increased, the
1
1
static in nature.
4304
Org. Lett., Vol. 11, No. 19, 2009

increased charged density resulted in higher I
Figure S5, Supporting Information).
Because of the extensive aggregation of compound 1 upon
E
/I
M
values
+ ssDNA-1) were incubated at 37.5 °C for 15 min.
Subsequent rapid cooling to -20 °C ensured that the mixture
containing a complementary sequence produced normal
duplex DNA, whereas the mutant could only give a mixture
of single stranded oligonucleotides. The addition of nuclease
S1 could completely remove all single strands in the reaction
mixtures, but had no effect on the duplex DNA.
As shown in Figure S10 (Supporting Information), the
mixture containing no mutation showed strong excimer
fluorescence after nuclease digestion. In contrast, excimer
fluorescence vanished with the mixture containing mis-
matched ssDNA-2. Mismatched sequences have a lower
duplex melting temperature. By careful selection of a suitable
annealing temperature, only the mixture containing the
complementary sequences could have duplex DNA. At the
same time, the mixture containing a single point mismatch
had little duplex formation. Mismatched ssDNAs cleaved
with nuclease S1 gave no induced pyrene probe aggregation,
so no excimer emission was detected.
(
binding to nucleic acid under our normal experimental
conditions, conventional methods for binding constant evalu-
ation could not be directly applied. However, under condi-
tions of lower compound 1 concentration, a good linear fit
was obtained. For binding of compound 1 to poly(dN)25, the
5
calculated binding constant was 1.6 × 10 . The correspond-
5
ing duplex gave a higher value of 3.1 × 10 , which is
consistent with the experimental results. In addition, Job’s
plot suggests that the binding stoichiometry between com-
pound 1 and the nucleic acid base of poly(dN)25 was about
1
.4:1. If pure electrostatic interactions were involved, we
would expect a binding stoichiometry of 1:1; thus, the results
suggest that in addition to ionic interactions, hydrophobic
interactions could also be involved in the binding process
(
see the Supporting Information).
As a proof-of-principle demonstration, we have utilized
the nucleic acid induced excimer fluorescence for the
detection of nuclease activity. A fixed amount of single-
strand specific nuclease S1 was mixed with poly(dN)25 [or
poly(dA)25] and incubated for a certain period of time.
Compound 1 was subsequently added, and the emission
spectrum was measured. The results show that strong excimer
emission was observed with 0 min incubation time, indicating
strong induced probe aggregation. Excimer emission gradu-
ally decreased with increasing incubation time and with no
induced excimer emission able to be observed after 25 min
of incubation (Figure S9, Supporting Information).
The results suggest that after prolonged incubation with
nuclease S1, more single-stranded DNA was hydrolyzed. As
mentioned above, little induced compound 1 aggregation
could be observed with an oligonucleotide sequence shorter
than five bases. Apparently, after degradation by nuclease
for a certain period of time, short oligonucleotides and
mononucleotides were produced, and the concentration of
oligonucleotides with a length greater than five bases
decreased. This resulted in a reduced degree of induced
aggregation of the probe molecule, and therefore, reduced
excimer emission was detected.
In conclusion, a positively charged pyrene probe (com-
pound 1) was synthesized. Oligonucleotides were shown to
induce aggregation of compound 1 via electrostatic and
hydrophobic interactions, and the induced π-π stacking
interactions between pyrene aromatic rings gave rise to strong
excimer emission. We demonstrate that the induced excimer
fluorescence is related to the concentration, length, sequence,
and the secondary structure of the nucleic acid, as well as
the concentration of the probe molecule. The induced excimer
emission was furthermore used to monitor the DNA cleavage
reaction by nuclease S1 and the detection of single-point
mutation in DNA. We believe that the properties of the probe
can be finely tuned, and that the assay conditions may be
further optimized. We envision that our method could assist
the future developments of basic biochemical research and
practical applications.
Acknowledgment. This work was supported by the
“Hundred Talents Project” (initiation support) of the Chinese
Academy of Sciences and the National Natural Science
Foundation of China (No. 20845006).
Supporting Information Available: Experimental details,
synthesis and characterization of compound 1, and UV-vis
and fluorescence studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
Pyrene excimer emission induced by nucleic acid can also
be applied to the detection of single nucleotide polymor-
phism. The target sequence mixed with the mutant sequence
(
ssDNA-0 + ssDNA-2) (Supporting Information) and the
target mixed with the complementary sequence (ssDNA-0
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