G-Tetraplex-Induced FRET within Telomeric Repeat Sequences Using PyA-PerA as Energy Donor-Acceptor Pair

Article abstract of DOI:10.1002/asia.201500996

G-tetraplex induced fluorescence resonance energy transfer (FRET) within telomeric repeat sequences has been studied using a nucleoside-tethered FRET pair embedded in the human telomeric G-quadruplex forming sequence (5′-A GGG TT^PyA GGG TT^PerA GGG TTA GGG-3′, Py=pyrene, Per=perylene). Conformational change from a single strand to an anti-parallel G-quadruplex leads to FRET from energy donor (^PyA) to acceptor (^PerA). The distance between the FRET donor/acceptor partners was controlled by changing the number of G-quartet spacer units. The FRET efficiency decreases with increase in G-quartet units. Overall findings indicate that this could be further used for the development of FRET-based sensing and measurement techniques.

Full text of DOI:10.1002/asia.201500996

DOI: 10.1002/asia.201500996
Communication
Fluorescence
G-Tetraplex-Induced FRET within Telomeric Repeat Sequences
Py
Using A-PerA as Energy Donor–Acceptor Pair
Rajen Kundu*[a]
two or more fluorophore units by controlling the number of
Abstract: G-tetraplex induced fluorescence resonance
energy transfer (FRET) within telomeric repeat sequences
nucleoside units.[13–18] During the last few years, several groups
have focused on FRET phenomena within the context of DNA
has been studied using a nucleoside-tethered FRET pair
to establish FRET engineering, to study DNA hybridization,
embedded in the human telomeric G-quadruplex forming
sequence (5’-A GGG TTPyA GGG TTPerA GGG TTA GGG-3’,
Py=pyrene, Per=perylene). Conformational change from
and/or for various biophysical studies of DNA.[14,19–24] However,
most of the efforts on DNA-mediated FRET processes have
been focused on duplex DNA systems or B-DNA with Watson–
a single strand to an anti-parallel G-quadruplex leads to
FRET from energy donor (PyA) to acceptor (PerA). The dis-
tance between the FRET donor/acceptor partners was
Crick base pairing. Very few reports have appeared in the liter-
ature[21–23] where non-B-DNAs, including G-quadruplexes, are
used to demonstrate FRET phenomena. It is noteworthy that
controlled by changing the number of G-quartet spacer
most of them contain fluorophores (or FRET pair) at the termi-
units. The FRET efficiency decreases with increase in G-
ni of the oligonucleotide sequence.[22–23] Thus, in such cases
quartet units. Overall findings indicate that this could be
the G-quartet spacer is not directly involved in the FRET engi-
further used for the development of FRET-based sensing
neering. To the best of our knowledge, till now, there is no
and measurement techniques.
report where fluorophores are covalently attached in the
middle (except the termini) of a G-quadruplex-forming oligonu-
cleotide sequence to study the engineering of tetraplex forma-
Fluorescence resonance energy transfer, so called FRET, is an
emerging technique widely used not only in materials science
but also in chemistry and biology.[1–11] FRET is a dipole–dipole
resonance interaction between two different fluorophores (say
donor and acceptor pair). When these donor–acceptor pairs
are placed within a proper distance (10–50 Š), FRET could be
possible from one fluorophore (called donor) to another fluo-
rophore (called acceptor) and thus, it is a distance-dependent
phenomenon.[12] The donor molecule of a FRET pair absorbs
the light (hn) at low wavelengths and emits a long wavelength
light. Meanwhile the acceptor molecule absorbs the long
wavelength light emitted from the donor molecule and emits
in the visible region (lem). Thus, FRET depends upon the spec-
tral overlap between the emission and absorption of the
donor and the acceptor, respectively.
tion and/or FRET. Thus, it is very interesting to construct such
a novel system and demonstrate FRET therein. This system can
be further used for the development of FRET-based detection
techniques.
Herein, the studies of G-tetraplex-induced fluorescence reso-
nance energy transfer within telomeric repeat sequences using
nucleoside-tethered FRET pairs embedded in the human telo-
meric G-quadruplex forming sequence 5’-A GGG TTA GGG TTA
GGG TTA GGG-3’ are reported. Telomere DNA sequences are
noncoding highly repetitive sequences that are present at the
termini of the eukaryotic chromosome.[25] Moreover, the activi-
ty of the human telomerase DNA depends upon the formation
and stabilization of their tetraplex structure. After careful inves-
tigation of the 3D structure of this quadruplex (PDB ID: 143D,
see Supporting Information),[26] the A7 and A13 positions were
chosen to place polycyclic aromatic fluorophores (or FRET pair)
almost parallel to the G-quartet plane (see Figure 1 and Sup-
porting Information). Moreover, these positions would not in-
terfere with the formation of the G-quadruplex structure and
also facilitate the stacking interaction between the fluorophore
and the external G-quartet. Polycyclic aromatic hydrocarbon,
pyrene (donor), and perylene (acceptor) chromophores are
well-established FRET pairs and have been widely used in bio-
physical studies of DNA by various groups.[14,24,27–31] These FRET
pairs were incorporated into the human telomeric sequence as
ethynylpyrene- and ethynylperylene-labelled 8-substituted 2’-
deoxyadenosine units (PyA and PerA, see Scheme 1 and Sup-
porting Information). Formation of a tetraplex/G-quadruplex
structure needs at least two G-quartet units. Thus, we can con-
DNA is considered as an ideal scaffold to demonstrate FRET
phenomena more accurately by changing the fluorophore-la-
belled base as well as by controlling the distance between the
[a] Dr. R. Kundu+
Department of Chemistry
Pohang University of Science and Technology
Pohang 790784 (South Korea)
E-mail: kundurajen@postech.ac.kr
[+] Present Address:
Department of Chemistry and Biochemistry
University of Colorado
Boulder 80303 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/asia.201500996.
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Communication
change from a single strand (“ss”) to an anti-parallel G-quadru-
plex (GQ).[32] Initially, circular dichroism (CD) spectroscopy was
performed to detect the conformational changes of the ODN
1, 2, and 3. The characteristic negative and positive CD bands
of G-quadruplex (GQ 1, 2, and 3) were identified at around
265 nm and 293 nm, respectively, after adding KCl (see Sup-
porting Information). This observation clearly indicates that the
incorporation of PyA and PerA do not interfere with the G-quad-
ruplex conformational stability.
Next, the UV-visible absorption experiments were carried out
with the ODN 1, 2, and 3 in the absence and presence of KCl
solution (Figure 2). Thus, the UV-visible spectra of single-strand
Figure 1. Schematic presentation of donor (PyA)/acceptor (PerA) tethered G-
quadruplex formation and G-quartet spacer-controlled fluorescence reso-
nance energy transfer (FRET).
Figure 2. UV-visible (chromophoric region) spectra of the donor/acceptor
tethered ODN 1, 2, and 3 in absence of KCl solution (single strand) and in
presence of KCl solution (G-quadruplex). All samples, [DNA]=1.5 mm,
[KCl]=400 mm, were prepared in 10 mm Tris- HCl buffer (pH 7.2) at 258C.
ODNs (in absence of KCl) showed strong absorption peaks at
around 385, 420, 455, and 485 nm in the chromophoric region.
After addition of KCl solutions to the ODNs to form the G-
quadruplex, the intensity of the absorbance peaks were dra-
matically decreased. Significant hypochromic shifts (ꢀ25%)
were observed in the chromophoric region of all the ODNs
after conformational change from a single strand to G-quadru-
plex. It is likely that this hypochromism is due to the hydro-
phobic stacking interaction between the fluorophores (pyrene
and perylene) and the external G-quartets.[33] Therefore, these
observations suggested that the PyA and PerA nucleoside units
placed the FRET pair in the favorable position with the external
G-quartets. The Tm values of GQ 1, 2, and 3 were slightly lower
(54.9, 56.8, 58.48C, respectively) than the corresponding un-
modified (absence of pyrene and perylene chromophores) G-
quadruplex ODNs (56, 57.8, and 59.58C, respectively). This ob-
servation showed that the presence of pyrene and perylene in
the middle of the ODNs slightly destabilized the G-quadruplex
structure.
Scheme 1. Chemical structure of FRET pair and FRET-pair-embedded G-quad-
ruplex-forming ODN sequences.
trol the distance of the FRET donor/acceptor partners by vary-
ing the number of G-quartet spacer units starting from 2, 3, 4,
etc. In this study, we have focused on G-quartet spacer units of
2, 3, and 4 (see Figure 1).
The fluorophore-labeled PyA- and PerA-phosphoramidite were
prepared starting from 2’-deoxyadenosine using literature
methods (see Supporting Information). Next, the PyA- and PerA-
phosphoramidite were incorporated at the A7 and A13 (start-
ing from 5’-end) positions of ODN 2 (5’-A (G)3 TTPyA (G)3 TTPerA
(G)3 TTA (G)3-3’), respectively, using an automated DNA synthe-
sizer. Accordingly, ODN 1 (5’-A (G)2 TTPyA (G)2 TTPerA (G)2 TTA
(G)2-3’), and ODN 3 (5’-A (G)4 TTPyA (G)4 TTPerA (G)4 TTA (G)4-3’)
were developed by decreasing and increasing of the number
of guanine nucleoside (G) units, respectively. The synthesized
ODNs were purified by HPLC and characterized by MALDI-TOF
mass spectrometry. Potassium chloride (KCl) solution was
added to the ODN 1, 2, and 3 to induce the conformational
After UV-visible study, fluorescence measurements were car-
ried out with the ODNs. Spectral overlap of PyA and PerA (see
Figure 3) satisfied the energy transfer from PyA from PerA. It was
assumed that in the single strand (unfolded) form, little or neg-
ligible energy transfer is expected from donor to acceptor as
the distance between two FRET partners is large.[34] Conforma-
tional change from a single strand to G-quadruplex upon treat-
ment with KCl solution leads to the FRET partners being in
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The efficiency of energy transfer (E), was calculated using
Equation (1):
F
F0
ð1Þ
E ¼ 1 À
where F and F0 are the fluorescence intensity of donor (pyrene
chromophore) in the tetraplex form (with the influence of ac-
ceptor) and in the single strand form (without the influence of
acceptor), respectively. The energy transfer efficiency (E) from
donor to acceptor was found as 66%, 55%, and 39% for ODN
1, 2, and 3, respectively. Thus, the FRET efficiency decreases
with increase in G-quartet units and ODN 3 showed the lowest
FRET efficiency among all three ODNs upon conformational
change from a single strand to an anti-parallel G-quadruplex.
The Fçrster radius (R0) for this system was calculated using
Equation (2):
Figure 3. Absorption and emission spectra of PyA and PerA chromophore.
Shaded area shows spectral overlap of FRET pair. Bold lines are extracted
from dotted lines. Blue, green, and red dotted lines denote absorption of
ssODN 1, emission of ssODN 1, and emission of GQ 1, respectively.
close proximity for energy transfer. The fluorescence spectra of
the ODNs exhibited two emission bands at around 450 and
520 nm for pyrene and perylene chromophores, respectively.
The excitation wavelength was chosen as 385 nm to measure
the fluorescence spectroscopy and thereby FRET. In the unfold-
ed single strand form, the emission band intensity at around
450 nm dominates over the emission band intensity at around
520 nm for ODN 1, 2, and 3. It is noteworthy that, in ODN 1,
the emission intensity at around 520 nm is slightly higher than
the other two ODN 2 and 3 and this may be because of little
energy transfer due to the smaller distance between donor
and acceptor (see Figures 1 and 4). Interesting fluorescence
1=6
R0 ¼ 0:2108½JðlÞk2nÀ4FDŠ
ð2Þ
where k2 is the orientation factor, n is the refractive index of
the medium, FD is the quantum yield of the donor in the ab-
sence of acceptor J(l) is the overlap integral of the fluores-
cence emission spectrum of the donor and the absorption
spectrum of the acceptor. Using the values of k2 =2/3, n=
1.33, FD =0.51, and the obtained overlap integral, J(l)=1.03”
1017, the Fçrster radius (R0) was obtained as 48.7 Š. Moreover,
the designed system showed that upon conformational
change from single strand to G-quadruplex structure leads to
the stacking interaction between the fluorophore and external
G-quartet. However, stacking interaction onto the external G-
quartet tends to slip, and thus the orientation of the FRET
pairs was not fully controlled during energy transfer in G-quad-
ruplex structure. Thus, the observed FRET of this designed
system might be due to both distant-dependent as well as ori-
entation-dependent phenomena.
In order to further gain insight in the FRET pair positions
and the interactions with the external G-quartet units upon
conformational change to a tetraplex/G-quadruplex structure,
MacroModel minimization was carried using AMBER* force
field.[35] Solution structure of the human telomeric G-quadru-
plex, PDB ID: 143D (5’-A GGG TTA GGG TTA GGG TTA GGG-3’)
was used for this calculation where A7 and A13 bases were re-
placed by PyA and PerA, respectively. The energy minimized
conformation of ODN 2 is shown in Figure 5. Thus, the Macro-
Model study showed that PyA placed the pyrene chromophore
parallel to the external G-quartet plane and stabilized through
hydrophobic stacking interaction with the G-quartet unit. In
contrast, due to the large size of the perylene unit, it resides
slightly outside from the G-quartet. However, PerA also placed
the perylene chromophore almost parallel to the G-quartet
plane.
Figure 4. Fluorescence emission spectra (lex =385 nm) of the donor/accept-
or tethered ODN 1, 2, and 3 in absence of KCl solution (single strand) and in
presence of KCl solution (G-quadruplex). All samples, [DNA]=1.5 mm,
[KCl]=400 mm, were prepared in 10 mm Tris- HCl buffer (pH 7.2) at 258C.
emission behavior was observed while adding KCl solution
into the ODNs. Decrease of emission intensity of the band at
450 nm and increase of emission intensity of the band at
520 nm were observed due to FRET for the ODN 1, 2, and 3
upon conformational change from a single strand to an anti-
parallel G-quadruplex. However, the decrease and increase of
emission intensities from the single strand to corresponding G-
quadruplex are not same for all the ODNs. ODN 1 contains two
G-quartet units; thus, smaller distance between FRET pair and
showed maximum decrease of emission intensity at 450 nm
and increase of emission intensity at 520 nm over ODN 2 and
3.
In conclusion, G-tetraplex-induced fluorescence resonance
energy transfer within telomeric repeat sequences was studied
using a nucleoside-tethered FRET pair embedded in the
human telomeric G-quadruplex forming sequence (5’-A GGG
TTPyA GGG TTPerA GGG TTA GGG-3’). Conformational change
from a single strand to an anti-parallel G-quadruplex upon
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Acknowledgements
The author is thankful to Pohang University of Science and
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Keywords: FRET · G-quadruplex · pyrene-perylene · telomere ·
tetraplex
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Manuscript received: September 16, 2015
Accepted Article published: October 22, 2015
Final Article published: November 4, 2015
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim