Dual door entry to exciplex emission in a chimeric DNA duplex containing non-nucleoside-nucleoside pair

Article abstract of DOI:10.1039/c3cc46967k

Dual door entry to exciplex formation was established in a chimeric DNA duplex wherein a fluorescent non-nucleosidic base surrogate (^OxoPyS) is paired against a fluorescent nucleosidic base surrogate ( ^TPhenB_Do). Packing of the nucleobases via intercalative stacking interactions led to an exciplex emission either via FRET from the donor ^TPhenB_Do or direct excitation of the FRET acceptor ^OxoPyS. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc46967k

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Dual door entry to exciplex emission in a chimeric
DNA duplex containing non-nucleoside–
nucleoside pair†
Cite this: Chem. Commun., 2014,
50, 829
Received 12th September 2013,
Accepted 22nd October 2013
Subhendu Sekhar Bag,*a Sangita Talukdar,a Rajen Kundu,a Isao Saitob and
Subhashis Janaa
DOI: 10.1039/c3cc46967k
www.rsc.org/chemcomm
Dual door entry to exciplex formation was established in a chimeric
DNA duplex wherein a fluorescent non-nucleosidic base surrogate
(
(
OxoPyS) is paired against a fluorescent nucleosidic base surrogate
TPhen
Do
B
). Packing of the nucleobases via intercalative stacking
interactions led to an exciplex emission either via FRET from the
TPhen
donor
B
Do
or direct excitation of the FRET acceptor OxoPyS.
¨
The phenomena of exciplex emission and Forster resonance
energy transfer (FRET) find widespread applications in chemis-
try, biology and material science.1,2 While FRET occurs in donor–
acceptor systems (D–A), an exciplex may form when the energy
levels of the donor and acceptor differ only by a few tenths of an
electron volt in such a D–A system. In the case of a spectral
overlap between the emission spectra of the acceptor and the
absorption spectra of the donor, energy transfer from the donor
Fig. 1 (a) Schematics of exciplex formation and the structures of the
acyclic non-nucleosidic base surrogate OxoPyS and the nucleosidic base
surrogate TPhenBDo. (b) The Amber* optimized geometry of the chimeric
base pair (OxoPyS:TPhenBDo) held by intercalative inter-strand p-stacking
interactions, showing the possibility of exciplex formation.
to the acceptor occurs. On the other hand, exciplex formation for hybridization is currently an attractive research area to study
involves complexation between the excited state of the donor/ exciplex formation and the FRET process in DNA for biophysical
acceptor and the ground state of the acceptor/donor, and is and material applications.
always characterized by a long radiative decay time and red-
Herein, we report a novel DNA hybridization system in which both
shifted fluorescence emission. FRET, being a distance depen- the exciplex as well as FRET emission were established when a
dent phenomenon, is being widely utilized in elucidating the fluorescent non-nucleosidic base surrogate, g-oxopyrenylserinol (OxoPyS),
structures and dynamics of DNA and proteins, in studying of an oligonucleotide probe paired against a fluorescent unnatural
biomolecular interactions, in DNA analysis and in many other nucleoside, triazolylphenanthrene (TPhenBDo),4 placed in a central posi-
sensory applications.1e,f,2 On the other hand, exciplex emission tion of a target oligodeoxynucleotide (ODN) to form a chimeric DNA
has received considerable attention due to its potential applica- duplex (Fig. 1). The logic behind choosing serinol as a non-ribose
tions in nucleic acid detection, and in biosensing and signaling acyclic scaffold was its more flexible and achiral nature compared to
devices in material science.1 To explore FRET interaction and other similar scaffolds used and its ability for insertion of a fluoro-
exciplex formation in DNA, multiple chromophore labeled oligo- phoric unit into DNA without affecting the duplex stability much.5 To
nucleotide probes have been developed for possible application the best of our knowledge, the design of a chimeric DNA duplex
in optical devices, light harvesting materials and in diagnostic wherein a fluorescent nucleosidic base surrogate paired against a non-
applications.3 Therefore, designing properly labeled DNA probes nucleosidic base surrogate is involved in a distance dependent energy
transfer process or exciplex formation is not known. Although, pyrene–
a Bio-organic Chemistry Laboratory, Department of Chemistry, Indian Institute of
Technology Guwahati, Guwahati-781039, Assam, India.
phenanthrene FRET/exciplex formation is known to some extent, an
oxopyrene fluorophore as a FRET acceptor is new.1b,c,g Moreover, FRET
was observed between the donor TPhenBDo nucleoside and the acceptor
OxoPyS non-nucleosidic base surrogate. Interestingly, we also observed
an exciplex emission at a longer wavelength than oxopyrene emission.
Our results indicated that a hybridization event in the chimeric DNA
E-mail: ssbag75@iitg.ernet.in; Fax: +91-361-258-2349
b NEWCAT Institute, School of Engineering, Nihon University, Koriyama, Fukushima
9638642, Japan
† Electronic supplementary information (ESI) available: Synthesis, characteriza-
tion, spectroscopic data. See DOI: 10.1039/c3cc46967k
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 829--832 | 829

View Article Online
Communication
ChemComm
dominant role in stabilizing a chimeric duplex containing OxoPyS
TPhen
paired against the hydrophobic nucleoside
B . The strong
Do
TPhen
pairing selectivity between OxoPyS and
B
Do
(compared to the
natural bases) and slightly higher stabilization (1.1 1C in Tm) of the
OxoPyS:TPhenBDo (ODN 1ꢀ2) duplex compared to that of a control A–T
pair could be rationalized through strong intercalative stacking
interactions between the oxopyrene unit of OxoPyS and the phenan-
threne moiety of TPhenBDo inside the duplex. However, the stability of
the most stable chimeric duplex (ODN 1ꢀ2) was found to be less by
3.2 1C compared to that of a natural C–G pair (Table 1).
Scheme 1 Synthesis of the acyclic non-nucleosidic base surrogate OxoPyS,
and the oligonucleotide probe ODN 1.
Next, we examined the UV-visible absorption properties of single
stranded ODN 1 and its various duplexes. While the duplexes of
OxoPyS with natural bases [ODN 1ꢀY (Y = A, G, C or T)] exhibited
duplex resulted in an exciplex emission, which was generated either via increased absorbance with a blue shift of the absorbance maxima (of
FRET or upon direct excitation of the FRET acceptor, oxopyrene.
about 3–6 nm), the chimeric duplex ODN 1ꢀ2 (OxoPyS:TPhenBDo) showed
The synthesis of the non-nucleosidic base surrogate OxoPyS and a bathochromic shift (B10 nm) in absorption wavelength with 20%
its incorporation into DNA is shown in Scheme 1. The synthesis of hypochromicity in oxopyrene absorbance in comparison to the
TPhen
the
B
Do
nucleoside and the DNA (ODN 2) was accomplished corresponding single stranded ODN 1 (ESI,† Fig. S3a and b). This
following our recently published protocol.4 Modified nucleotides observation indicated that the oxopyrene unit of the non-nucleosidic
were placed at the centre of each palindromic DNA sequence base surrogate OxoPyS is involved in strong intercalative stacking
(Table 1). Incorporation of the amidite 6 generated two isomeric interactions inside the chimeric DNA duplex.6 Thus, there is a chance
oligodeoxynucleotides (ODNs), most likely two rotamers, containing of p–p stacking interactions between the chromophoric moieties of
OxoPyS (ESI,† Fig. S1). The isomer with longer retention time (ODN 1) two chimeric bases inside the duplex, which is also supported by the
was used in this study because of the comparatively better discri- high thermal melting stability of the chimeric duplex (Table 1).
minating fluorescence signal achieved in sensing the presence of
the opposite natural base of a target DNA.
The fluorescence photophysical properties revealed that upon
excitation at the absorbance maximum of OxoPyS (365 nm), all the
The study of the thermal stability of various duplexes showed that duplexes with natural nucleosides, except guanosine, showed strong
the stability of the chimeric duplex ODN 1ꢀ2 (52.3 1C) was comparable fluorescence emission compared to the single strand ODN 1. The
to that of a natural A–T pair (51.2 1C). It was also evident that the duplex ODN 1ꢀ4, wherein OxoPyS is paired against the strong electron
chimeric duplex ODN 1ꢀ2 was more stable than the duplexes with any donor guanosine, exhibited strongly quenched emission. The emis-
of the natural bases (Table 1). Moreover, the acyclic non-nucleosidic sion maxima in all cases was centered at B452 nm with a tail
base surrogate OxoPyS showed significant selectivity (1.9–3.9 1C higher expanding up to 575 nm. Surprisingly, the chimeric duplex ODN 1ꢀ2
TPhen
in Tm stability) for the nucleosidic base surrogate
B
Do
over all showed a strong and broad emission band centered at 460–535 nm
four natural bases (Table 1). These results showed that strong that spanned up to 675 nm (Fig. 2a). The long tail and broad shape of
hydrophobic and p–p stacking interactions may have played a the emission prompted us to re-examine and resolve the spectra.
We could clearly resolve the broad spectra, which indicated dual
emission – the pure oxopyrene monomer emission at 460 nm and
TPhen
Table
1
Oligonucleotide sequences containing OxoPyS and
B
Do
another intense emission centered at 535 nm and spanning up to
675 nm (Fig. 2b). The intense and broad long wavelength emission
was most likely from the exciplex of OxoPy–TPhen. As the oxopyrene
moiety is involved in intercalative stacking interactions inside the
nucleobase surrogates and their natural complements, and the photo-
physical and thermal properties of the various ODNsa
ODN
Sequence
ODN 1
ODN 2
ODN 3–6
50-CGCAAT OxoPyS TAACGC-30
TPhen
30-GCGTTA
B
ATTGCG-50
Do
30-GCGTTA N ATTGCG-50 [N = A, G, C, T]
ODN labs (nm) lfl (nm) Ff
Tm (1C) t1 (ns) t2 (ns) hti (ns)
1
362
452
0.24
—
1.6
2.1
7.5
2.2
1.4
2.4
2.6
4.1
7.6
14.8
5.1
4.4
5.3
5.4
2.8
6.0
13.9
3.7
2.5
3.8
3.5
1ꢀ2b 372
460, 535 0.46c 52.3
1ꢀ3
1ꢀ4
1ꢀ5
1ꢀ6
357
358
356
359
455
452
454
453
0.41 48.4
0.13 48.5
0.39 50.4
0.44 49.2
Fig. 2 (a) Emission spectra (lex = 365 nm) of ODN 1 and its various duplex
a
The Tm of natural AꢀT and CꢀG pairs are 51.2 1C and 55.5 1C,
respectively. The error in Tm is estimated at ꢁ 0.3 1C. All life times
ODNs. (b) Normalized variable temperature fluorescence emission (lex
=
b
365 nm) of the chimeric duplex ODN 1ꢀ2 (multi gauss fit) showing evidence
of exciplex formation. Each single stranded ODN was 1.0 mM in 50 mM
sodium phosphate, 0.1 M sodium chloride, pH 7.0.
were measured using lex = 375 nm; lem = 460 nm and for ODN 1ꢀ2, in
c
addition 535 nm (second row), were used to monitor. The monomer
and exciplex quantum yields are 0.18 and 0.28, respectively.
830 | Chem. Commun., 2014, 50, 829--832
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
TPhen
duplex DNA, there is a high possibility of formation of a p–p stacked due to FRET from the
B
Do
donor, we would observe only
complex between the excited oxopyrene and the ground state of one emission band at around 460 nm. However, we observed a
triazolyl phenanthrene. To prove our theory, we recorded variable broad emission with two resolvable bands at 460 nm (the char-
temperature emission spectra maintaining the same conditions of acteristic emission of the OxoPy monomer) and at 535 nm, which
excitation. Thus, we observed that as the temperature was increased, could clearly be assigned as OxoPy–TPhen exciplex emission (Fig. 3b
the emission intensity at 535 nm decreased, indicating the break- and ESI,† Fig. S7c). Therefore, it was clear from the emission spectra
down of the p-stacked excited state complex (exciplex) between OxoPy that upon excitation at 310 nm, the excited triazolylphenanthrene
and TPhen (Fig. 2b and ESI,† Fig. S7a).1b,c
Therefore, it was clear that excitation (at 365 nm) of the of OxoPyS paired against
transferred its energy to the closely spaced oxopyrene chromophore
TPhen
B
Do
in the chimeric duplex ODN 1ꢀ2.
oxopyrene moiety of OxoPyS in the chimeric duplex gave rise to the Absorbing the transferred energy (FRET energy), the OxoPy chro-
generation of OxoPy–TPhen exciplex emission at around 535 nm, mophore went to the excited state and formed a p-stacked complex
along with the OxoPy monomer emission at 460 nm. That the (exciplex) with the relaxed TPhen chromophore, which then released
exciplex was formed in a ground state complex in a p–p stacked its energy in the form of emission light at 535 nm. We calculated the
¨
fashion was also evident from excitonic interactions seen in the Forster distance (R0) to be 22 Å with a FRET efficiency of 43% (ESI,†
absorption spectra (ESI,† Fig. S3a and b). Time resolved fluores- Section S10). The variable temperature emission spectra showed a
cence at 25 1C and at higher temperatures (lex = 375 nm) with the gradual decrease in emission intensity of the 535 nm band and an
chimeric duplex ODN 1ꢀ2 revealed that as the temperature increase in the intensity of the donor emission band as the
increases, the % composition and life time of the component with temperature was increased (Fig. 3b). This observation indicated
longer life time was found to decrease drastically (Table 1 and ESI,† exciplex formation via FRET. That the exciplex was formed in a
Fig. S8). This observation also corroborated with exciplex emission. ground state complex in a p–p stacked fashion was also evident
After investigating the exciplex emission from the chimeric from excitonic interactions seen in the absorption spectra (ESI,†
duplex ODN 1ꢀ2, we turned our attention to examine the possibility Fig. S3a and b). Formation of the exciplex was also supported from
of a FRET interaction between the two fluorophoric base surrogates. the Amber* optimized geometry of the chimeric duplex (Fig. 1b).7
The primary condition for FRET to occur was satisfied by the overlap
In summary, this newly designed chimeric DNA duplex repre-
TPhen
of the emission spectra of the
B
Do
containing ODN 2 (donor) sents a very interesting dual door entry system for exciplex emission.
with the absorption spectra of the OxoPyS containing ODN 1 (accep- All the observations indicated that both processes of excitation of
tor) (ESI,† Fig. S9).2 It was observed that the fluorescence intensity of the oxopyrene chromophore of OxoPyS – either energy transfer from
the acceptor OxoPyS in the chimeric duplex ODN 1ꢀ2 increased from the excited triazolylphenanthrene (FRET) to oxopyrene or direct
the emission in single stranded ODN 1 by almost two times in the excitation of OxoPyS at its absorbance maximum – led to exciplex
presence of the donor TPhenBDo in duplex ODN 1ꢀ2, when excited at emission. Design of such systems would have great impact in
TPhen
the absorption maximum of
B
Do
(lmax = 310 nm, wherein devising optoelectronics and might find applications in chemistry,
negligible absorbance was there for OxoPyS). On the other hand, the biology, material sciences and in diagnostic technology.
fluorescence intensity of the donor TPhenBDo in the chimeric duplex
This work was financially supported by the Department of Science
ODN 1ꢀ2 decreased compared to the emission in the single stranded and Technology [DST: SR/SI/OC-69/2008], Govt. of India. Sincere thanks
ODN 2. This change in fluorescence intensity revealed visual to Prof. P. Phukan, Guahati University for recording the NMR spectra.
evidence of a FRET process from TPhenBDo to OxoPyS (Fig. 3a).2f
However, upon excitation of the duplex ODN 1ꢀ2 at the absor-
bance maximum of the donor TPhenBDo (lex = 310 nm), surprisingly,
Notes and references
1 (a) J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer,
a broad and intense emission was observed. This emission was
superimposable to the spectrum that was observed when excited at
New York, 3rd edn, 2006; (b) I. Trkulja and R. Haner, J. Am. Chem.
the absorbance maximum (365 nm) of the acceptor, OxoPyS. If the
Soc., 2007, 129, 7982; (c) I. Trkulja and R. Haner, Bioconjugate Chem.,
2007, 18, 289; (d) M. Mac, T. Uchacz, A. Danel, M. A. Miranda, C. Paris
and U. Pischel, Photochem. Photobiol. Sci., 2008, 7, 633; (e) Y. N. Teo
and E. T. Kool, Bioconjugate Chem., 2009, 20, 2371; ( f ) Y. N. Teo,
J. N. Wilson and E. T. Kool, J. Am. Chem. Soc., 2009, 131, 3923;
(g) F. Garo and R. Haner, Angew. Chem., Int. Ed., 2012, 51, 916.
emission was only the emission from the oxopyrene chromophore
¨
2 (a) T. Forster, Naturwissenschaften, 1946, 6, 166; (b) A. Iqbal, S. Arslan,
B. Okumus, T. J. Wilson, G. Giraud, D. G. Norman, T. Ha and D. M. J.
Lilley, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 11176; (c) B. Juskowiak, Anal.
Bioanal. Chem., 2011, 399, 3157; (d) S. Preus and L. M. Wilhelmsson,
ChemBioChem, 2012, 13, 1990; (e) T. Kato, H. Kashida, H. Kishida, H. Yada,
H. Okamoto and H. Asanuma, J. Am. Chem. Soc., 2013, 135, 741; ( f ) R. J.
Nedumparaa, P. J. Manua, C. P. G. Vallabhanb, V. P. N. Nampooria and
P. Radhakrishnana, Opt. Laser Technol., 2008, 40, 953.
3 (a) V. L. Malinovskii, F. Samain and R. Haner, Angew. Chem., Int. Ed., 2007,
46, 4464; (b) E. Schwartz, S. Le Gac, J. J. L. M. Cornelissen, R. J. M. Nolte
and A. E. Rowan, Chem. Soc. Rev., 2010, 39, 1576; (c) V. L. Malinovskii,
D. Wenger and R. Haner, Chem. Soc. Rev., 2010, 39, 410; (d) Y. Krishnan
and F. C. Simmel, Angew. Chem., Int. Ed., 2011, 50, 3124; (e) T. J. Bandy,
A. Brewer, J. R. Burns, G. Marth, T. Nguyen and E. Stulz, Chem. Soc. Rev.,
2011, 40, 138; ( f ) Y. N. Teo and E. T. Kool, Chem. Rev., 2012, 112, 4221.
Fig. 3 (a) Fluorescence emission spectra (lex = 310 nm) of ODN 1, ODN 2
and chimeric duplex ODN 1ꢀ2. (b) Normalized variable temperature
fluorescence emission spectra of chimeric duplex ODN 1ꢀ2 (lex = 310)
(multi gauss fit) showing evidence of exciplex formation.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 829--832 | 831

View Article Online
Communication
ChemComm
4 S. S. Bag, S. Talukdar, K. Matsumoto and R. Kundu, J. Org. Chem.,
2013, 78, 278.
(d) H. Kashida, K. Murayama, T. Toda and H. Asanuma, Angew.
Chem., Int. Ed., 2011, 50, 1285.
5 (a) B. N. Trawick, T. A. Osiek and J. K. Bashkin, Bioconjugate Chem., 6 (a) G. Dougherty and J. R. Pilbrow, Int. J. Biochem. Cell Biol., 1984, 16, 1179;
2001, 12, 900; (b) H. Kashida, X. Liang and H. Asanuma, Curr. Org.
Chem., 2009, 13, 1065; (c) H. Asanuma, T. Toda, K. Murayama,
X. Liang and H. Kashida, J. Am. Chem. Soc., 2010, 132, 14702; 7 Maestro, version 9.0, Schrodinger, LLC, New York, NY, 2009.
(b) M. Nakamura, Y. Fukunaga, K. Sasa, Y. Ohtoshi, K. Kanaori,
H. Hayashi, H. Nakano and K. Yamana, Nucleic Acids Res., 2005, 33, 5887.
¨
832 | Chem. Commun., 2014, 50, 829--832
This journal is ©The Royal Society of Chemistry 2014