DNA mimics of red fluorescent proteins (RFP) based on G-quadruplex-confined synthetic RFP chromophores

Article abstract of DOI:10.1093/nar/gkx803

Red fluorescent proteins (RFPs) have emerged as valuable biological markers for biomolecule imaging in living systems. Developing artificial fluorogenic systems that mimic RFPs remains an unmet challenge. Here, we describe the design and synthesis of six

Full text of DOI:10.1093/nar/gkx803

10380–10392 Nucleic Acids Research, 2017, Vol. 45, No. 18
Published online 13 September 2017
doi: 10.1093/nar/gkx803
DNA mimics of red fluorescent proteins (RFP) based
on G-quadruplex-confined synthetic RFP
chromophores
Guangfu Feng1, Chao Luo1, Haibo Yi1, Lin Yuan1, Bin Lin2, Xingyu Luo1, Xiaoxiao Hu3,
Honghui Wang4, Chunyang Lei1, Zhou Nie1,* and Shouzhuo Yao1
1State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering,
Hunan University, Changsha 410082, PR China, 2Pharmaceutical Engineering & Key Laboratory of Structure-Based
Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China,
3Molecular Science and Biomedicine laboratory, Hunan University, Changsha 410082, PR China and 4College of
Biology, Hunan University, Changsha 410082, PR China
Received July 13, 2017; Revised August 26, 2017; Editorial Decision August 28, 2017; Accepted August 31, 2017
ABSTRACT
INTRODUCTION
Green fluorescent proteins (GFP) are essential tools in
biomedical and pharmacologic studies due to their genetic
encoding properties and intrinsic high fluorescence quan-
tum yields (1–3). When fused with proteins of interest, they
can be utilized as a fluorescent marker suitable for visu-
alizing protein localization and biochemical trafficking in
live cells (4). Red fluorescent proteins (RFP) have, to date,
received more attention than GFPs as they are indispens-
able complements to the FP spectral palette and have a
number of advantages in bio-imaging. RFPs exhibit fluores-
cence emission maxima beyond 570 nm, thus they have less
autofluorescence interference of cells, lower light-scattering,
and lower photo-toxicity (5). These make RFPs especially
valuable for deep tissue and whole body imaging. RFPs
have a chromophore similar to the p-hydroxybenzylidine-
imidazolinone (HBI) core of GFP, except that it possesses
an additional N-acylimine substituent on the HBI motif.
The acylimine extends the ␲-electron delocalized network
of HBI and results in a red-shift in absorption and emis-
sion relative to GFP (6). The RFP protective ␤-barrel struc-
ture provides a rigid and hydrophobic environment which
confines a coplanar chromophore structure, and ensures the
emission pathway dominant in excited state energy release.
Developing synthetic materials mimicking natural flu-
orescent proteins is crucial for elucidating photo-physical
mechanism of FPs and creating new emission-responsive
materials and biosensors. Unfortunately, most of synthetic
chromophores of RFP are non-fluorescent similar to de-
natured RFP, owing to ultrafast intramolecular vibration
(7,8). Developing highly emissive molecular mimics of RFP
is still challenging. Currently, only two cases of emissive
RFP analogues with decent quantum yield (>0.02) are
available based on confinement of RFP chromophore ana-
Red fluorescent proteins (RFPs) have emerged as
valuable biological markers for biomolecule imag-
ing in living systems. Developing artificial fluoro-
genic systems that mimic RFPs remains an unmet
challenge. Here, we describe the design and syn-
thesis of six new chromophores analogous to the
chromophores in RFPs. We demonstrate, for the
first time, that encapsulating RFP chromophore ana-
logues in canonical DNA G-quadruplexes (G4) can
activate bright fluorescence spanning red and far-
red spectral regions (Em = 583−668 nm) that nearly
match the entire RFP palette. Theoretical calcula-
tions and molecular dynamics simulations reveal
that DNA G4 greatly restricts radiationless deacti-
vation of chromophores induced by a twisted in-
tramolecular charge transfer (TICT). These DNA mim-
ics of RFP exhibit attractive photophysical proper-
ties comparable or superior to natural RFPs, includ-
ing high quantum yield, large Stokes shifts, excellent
anti-photobleaching properties, and two-photon flu-
orescence. Moreover, these RFP chromophore ana-
logues are a novel and distinctive type of topology-
selective G4 probe specific to parallel G4 conforma-
tion. The DNA mimics of RFP have been further ex-
ploited for imaging of target proteins. Using cancer-
specific cell membrane biomarkers as targets, long-
term real-time monitoring in single live cell and two-
photon fluorescence imaging in tissue sections have
been achieved without the need for genetic coding.
*To whom correspondence should be addressed. Tel: +86 731 88821626; Fax: +86 731 88821848; Email: niezhou.hnu@gmail.com
ꢀ
C
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Nucleic Acids Research, 2017, Vol. 45, No. 18 10381
logues via covalent structure locking or the assembly of
chromophores with proteins (9,10). Given the significance
of RFP in in vivo imaging, the scarcity of fluorescent RFP
mimics greatly restricts the available spectral region of syn-
thetic FP-mimicking materials and their use in multi-color
or deep-tissue imaging.
logues perform as ‘smart’ G-quadruplex ligands having the
dual-functionalities of being potent structural stabilizers
and topology-selective fluorescent sensors capable of recog-
nizing parallel G4 against other G4 or DNA conformations.
These DNA RFP mimics can be further exploited to ‘role-
play’ as natural RFPs for imaging of target proteins. Both
one-photon and two-photon fluorescent imaging of cancer-
specific cell membrane biomarkers, such as protein tyrosine
kinase-7 (PTK7), has been achieved in a live cell and tissue
setting without genetic coding being required.
Unlike the largely unexplored fluorescent RFP mimics,
emissive GFP mimics have been extensively studied. A
number of approaches have achieved emissive GFP ana-
logues via mimicking the natural confined effect of GFP
␤-barrel to restrict the intramolecular motions of GFP
chromophores. They include: structure locking by chemi-
cal linkage (11–16), both covalent and non-covalent; and,
GFP chromophore encapsulation into a rigid environ-
ment, such as supra-molecular hosts (17,18), metal-organic
frameworks (19), the framework of aggregation-induced
emission (AIE) (20,21), protein assembly (22,23), and ri-
bonucleic acid (RNA) aptamers. A fluorogenic RNA ap-
tamer ‘Spinach’ developed by Jaffrey et al. is an intrigu-
ing GFP mimic with strong emission comparable to na-
tive GFPs (24–26), which was selected by the systematic
evolution of ligands by exponential enrichment (SELEX)
process. Its crystal structures demonstrated that ‘Spinach’
contain a unique G-quadruplex (G4)-containing binding
pocket, which is composed of a non-canonical G4 RNA
module, an unpaired G, and an adjacent base-triplet, to ac-
commodate GFP chromophore analogues (27,28). This sig-
nificant finding implied the potential of G-quadruplex as a
promising structural motif for construction of fluorogenic
GFP mimics. However, several important issues still need
to be addressed: (i) except the selected G4-containing RNA
aptamers, whether canonical G4 structures enable fluores-
cent activation of FP chromophores have not been inves-
tigated; (ii) given the polymorphism of G4, the impact of
G4 topology on its ability to activate FP mimics is also un-
explored; (iii) most relevantly, except GFP chromophores
analogues, whether RFP chromophores could be fluores-
cently activated by G4 or other nucleic acid structures is
totally unknown.
Herein, we report the design and synthesis of a set of
new multi-color chromophores resembling the RFP chro-
mophores (Scheme 1). We prove, for the first time, that RFP
chromophore analogues are a new type of G-quadruplexes
ligand and encapsulating RFP chromophore analogues in
canonical DNA G4s can elicit bright fluorescence span-
ning red and far-red spectral regions (Em = 583−668 nm),
which perfectly match existing RFP palettes. G-quadruplex
is an alternative secondary nucleic acid structure formed by
guanine-rich (G-rich) DNA sequences. The G4 G-tetrad,
which is formed by a four guanine via Hoogsteen hydrogen-
bonding, provides an ideal docking site for RFP chro-
mophore analogue stacking and restricts them to a pla-
nar conformation for efficient emission, which resemble the
confinement of natural RFP ␤-barrel structure (Scheme
1A). These unique DNA RFP mimics have several photo-
physical merits, such as high quantum yields (maximum =
0.39), large Stokes shifts (maximum = 112 nm), and su-
perior anti-photobleaching properties. It is worth to note
that the G4-confined RFP chromophores exhibit unique
two-photon fluorescence and holds promise for live systems
imaging. Moreover, the proposed RFP chromophore ana-
MATERIALS AND METHODS
Unless otherwise stated, all the organic reagents were pur-
chased from commercial suppliers and used without further
purification. THF were dried and purified by distillation
from sodium/benzophenone and CaH2. Tris, KCl, EDTA
and MgCl2 were purchased from Sangon Biotech (Shang-
hai, China). DPBS, penicillin, streptomycin, RPMI 1640,
McCoy’s 5a Medium Modified and fetal bovine serum were
obtained from Thermo Scientific (USA). Ultrapure water
(18.2 Mꢀ·cm) from the Millipore Milli-Q system was used
in the experiments.
NMR spectra were recorded on a Bruker-400 spectrome-
ter, using TMS as an internal standard. Chemical shifts (␦)
are quoted in parts per million (ppm) and coupling con-
stant (J) are recorded in Hertz (Hz). Mass spectra were
performed using an LCQ Advantage ion trap mass spec-
trometer from Thermo Finnigan. All reactions were mon-
itored by TLC with Macherey-Nagel pre-coated Glassic
sheets (0.20 mm with fluorescent indicator UV254). Com-
pounds were visualized with UV light at 254 nm and 365
nm. Flash column chromatography was carried out using
silica gel from Merck (200–300 mesh). The pH measure-
ments were carried out on a Mettler-Toledo Delta 320 pH
meter. Fluorescence lifetime measurements were performed
on a FLS980 fluorescence spectrofluorometer (Edinburgh
Instruments, USA).
Oligonucleotides (HPLC-purified, sequences were shown
in Supplementary Tables S1 and S6) were synthesized and
purified by Sangon Biotech (Shanghai, China). Oligonu-
cleotides were dissolved in buffer solution (25 mM Tris and
pH 8.0) at 100 ␮M and annealed (heated to 95◦C for 10
min and then cooled slowly to 4◦C) prior to be used in each
experiment. The dsDNA was prepared by annealing equal
amounts of complementary strands in buffer solution (25
mM Tris, 10 mM MgCl2 and pH 8.0).
Circular dichroism (CD) spectroscopy
The CD spectra were recorded on MOS-500 spectropho-
tomer (BioLogic, France) at room temperature (RT, 25◦C).
Before the CD measurement, the oligonucleotides were an-
nealed following the above-mentioned conditions prior to
measurements. For CD measurements, samples were trans-
ferred into 2 mm path length quartz cuvettes, scanning from
220 nm to 360 nm at a rate of 50 nm/min for three times
with the slit of 5 nm. CD melting curve was performed in
the temperature range of 24–96◦C at an interval of 2.5◦C
with a heating rate of 1◦C/min.

10382 Nucleic Acids Research, 2017, Vol. 45, No. 18
Scheme 1. (A) Structures of a RFP (upper) and a DNA mimic of RFP composed of a DNA G-quadruplex and a synthetic RFP chromophore analogue
(bottom). Chromophores are emphasized in red. (B) Chemical structures of the chromophores in RFPs. (C) Structures of the RFP chromophore analogues
synthesized in this study. (D) Synthetic route of the RFP chromophore analogues using the following reagents and conditions: a) hexamethylenetetramine,
TFA, 120◦C, 12 h; b) N-Acetylglycine, anhydrous sodium acetate, acetic anhydride, 120◦C, 4 h; c) 40% aq. CH3NH2, K2CO3, EtOH, 80◦C, 4 h; and d)
ZnCl2, THF, RCHO, 80◦C.
Ultraviolet–visible (UV–Vis) spectroscopy
native electrophoresis using a 20% acrylamide gel, stained
with DFHBFSI (2 ␮M) in Tris–HCl buffer (25 mM Tris,
100 mM KCl, 50 mM MgCl2 and pH 8.0) and visualized via
fluorescence detection using a ChemiDoc MP system (Bio-
Rad, USA).
Please see supporting information (SI) for the experi-
mental details of measurement of two-photon cross sec-
tion, pKa measurements, quantum yield calculations, disso-
ciation constant measurements, photo-bleaching analysis,
molecular modelling, DFT calculations, cell culture, cyto-
toxicity measurements, construction of plasmids and real-
time quantitative reverse transcription PCR, flow cytom-
etry and confocal laser scanning microscopy assay, tissue
imaging.
UV–Vis spectra were recorded on a Varian Cary 100-Bio at
RT using a 10-mm path length quartz cuvette. For titration
experiment, small aliquots of a stock solution of oligonu-
cleotide were added into the solution containing DFHBFSI
(10 ␮M) in Tris-HCl buffer (25 mM Tris, 100 mM KCl,
50 mM MgCl2 and pH 8.0). The final concentrations of
oligonucleotide were varied from 0 to 15 ␮M. The absorp-
tion spectra from 400 nm to 700 nm were measured after
incubation at RT for 20 min.
Fluorescence Spectroscopy and Real-time Fluorescent
Monitoring the Topological Switch of G4. Fluorescence ex-
periments were carried out using a QuantaMaster™ fluo-
rescence spectrophotometer (PTI, Canada). A quartz cu-
vette with 2 mm × 10 mm path length was used for spec-
trum measurements, and both excitation and emission slit
widths were 10 nm unless otherwise specified. For fluores-
cence titration experiments, DFHBFSI (3 ␮M) and pre-
annealed oligonucleotides (0–15 ␮M) were incubated for 20
min in Tris–HCl buffer (25 mM Tris, 100 mM KCl, 50 mM
MgCl2 and pH 8.0). For real-time fluorescent monitoring,
the topological switch of G4, DFHBFSI (1 ␮M) and PW17
(G-quadruplex, 1 ␮M) were incubated in 100 ␮l of Tris–
HCl buffer (25 mM Tris, 2 mM KCl, 50 mM MgCl2 and
pH 8.0) for 5 min. Then, 3 ␮l of 5 mM Pb2+ was added into
the solution. Ten minutes later, 3 ␮l of 5 mM EDTA was
added into the solution. During these process, fluorescence
intensity at 606 nm was monitoring with an excitation wave-
length of 513 nm on the fluorescence spectrometer.
RESULTS AND DISCUSSION
G-quadruplex-chromophore complexes as DNA RFP mimics
Inspired by that RNA aptamer ‘Spinach’ contains a com-
posite binding pocket with a non-canonical G4 module to
accommodate the GFP chromophore analogue DFHBI, an
optimized derivative of HBI (27,28), we thus tested whether
a canonical DNA G4 structure could elicit the fluores-
cence of synthetic FP chromophores. Given that both GFP
and RFP share the common HBI core, we first examined
the influence of G4 structures on the fluorescence of HBI
and its analogue DFHBI prior to investigating RFP chro-
mophores. HBI and DFHBI were prepared via an Erlen-
meyer azlactone synthesis (15,24) and treated with a known
G4-forming DNA sequence NG16 in the presence of 100
mM K+ ion. The sequence of NG16 is shown in Supplemen-
tary Table S1, and the CD characterization of its G4 confor-
mation is described below. As shown in Figure 1A, NG16
shows negligible influence on the absorption properties of
Gel electrophoresis studies
Twenty microliters of NG16 at various concentrations or
different G4 oligonucleotides were loaded and separated by

Nucleic Acids Research, 2017, Vol. 45, No. 18 10383
Figure 1. (A) Absorption spectra and Q.Y. of HBI (3 ␮M) and DFHBI (3 ␮M) in the absence and presence of NG16 (30 ␮M). (B) NG16 (15 ␮M) enhances
the fluorescence of HBSI (3 ␮M) and HBNI (3 ␮M). Inset is the photograph of HBSI and HBNI with NG16 under illumination of ultraviolet (UV, 365
nm) light. (C) Structures of the RFP chromophore analogues. (D) Absorption, (E) excitation and (F) emission spectra of chromophore–NG16 complexes
(3 ␮M). Inset of F is the solutions containing DFHBFSI, DFHBFSI with NG16, and DFHBFSI with calf thymus DNA (CtDNA) photographed under
illumination of UV light. (G) Chromophore-NG16 complexes were illuminated with UV light and photographed. The image is a montage obtained under
identical image-acquisition conditions.
HBI/DFHBI and can’t activate their fluorescence even at
considerably high stoichiometric ratio (50 eq., Supplemen-
tary Table S2). Similar results were also observed for other
G4-forming sequences (Supplementary Table S3). Hence,
conventional G4 structures alone, without any other addi-
tional specific contacts as found in spinach, are not efficient
for GFP chromophores binding and fluorescent activation.
This might result from that the p-aromatic surface of HBI
analogues is relative small thus unable to interact with a ter-
minal G-quartet strongly via ␲–␲ stacking.
We envisaged that the RFP chromophores, especially
Kaede-like chromophores (Scheme 1B), bearing additional
aromatic ring conjugated with HBI core would lead to two
desirable features: one is the spectroscopic red-shift com-
pared with GFP chromophores due to extended conju-
gation; the other is the strengthened interaction with G-
quartet of G4 structure by the chromophores’ enlarged

10384 Nucleic Acids Research, 2017, Vol. 45, No. 18
Unique photo-physical properties of DNA RFP mimics
␲-ring system. To test this assumption, we have synthe-
sized two RFP chromophores with aromatic substituents
linking to HBI core via a vinyl group according to a lit-
erature procedure (29), termed HBSI and HBNI (Figure
1B). The theoretical prediction was performed by the on-
line molecular docking system of the G-quadruplex Lig-
ands Database (G4LDB) (30), and the results suggested
that HBSI and HBNI are preferable ligands of G4 com-
pared to HBI and DFHBI due to their additional ␲-ring
(Supplementary Figure S1). Inspiringly, the experimental
data (Figure 1B) showed that HBSI and HBNI fluorescence
markedly increased by 66.8 and 68.5 folds, respectively,
while bound with NG16, which is closely consistent with the
theoretical predictions. This indicates that these RFP chro-
mophores are fluorescently activatable through their bind-
ing with a DNA G4 structure. However, a limitation is that
they fluoresce green and purplish green with emission max-
ima at 505 and 537 nm, respectively, which are significantly
short than those of RFPs (>570 nm). An analysis of ab-
sorption properties of HBSI and HBNI indicated that their
phenol form (protonated) was predominant at neutral pH
(Supplementary Figure S2). This is consistent with their rel-
ative high pKa (the dissociation constant of phenolic OH
group) which are 8.8 and 9.3 for HBSI and HBNI, respec-
tively (Supplementary Figure S3). Supplementary Figure
S2 shows that the chromophores in 0.1 M NaOH exhibit
remarkable absorption bathochromic shifts (∼49 nm and
∼52 nm for HBSI and HBNI). This indicates their pheno-
late forms are more relevant for mimicking RFPs, which
is in accordance with natural chromophores in RFPs and
other synthetic ones (6,29).
To obtain G4-chromophore complexes with red emis-
sion comparable with RFPs, we sought to further adjust
spectroscopic red-shift of chromophores via introducing
electron withdrawing substituents to the phenolic ring to
facilitate its deprotonation at neutral pH. Two new RFP
chromophores with fluorine substituents, DFHBSI and
DFHBNI (Figure 1C), were synthesized from the conden-
sation of DFHBI with corresponding arylaldehydes cat-
alyzed by ZnCl2 (Scheme 1D). As expected, their pheno-
lic acidities were obviously enhanced (pKa = 6.4 and 7.3
for DFHBSI and DFHBNI, Supplementary Figure S3)
compared to their unsubstituent counterparts, HBSI and
HBNI. Correspondingly, the chromophore-NG16 com-
plexes of DFHBSI and DFHBNI exhibited a bright red
fluorescence with a single emission at 590 and 612 nm
(Figure 1D–G), respectively, which is identical to conven-
tional RFPs (Supplementary Table S4). Furthermore, to
determine whether the DNA RFP mimics could be spec-
trally modulated by the diverse chromophores, another
four novel RFP chromophores were generated by introduc-
ing either electron-donating (methoxyl or dimethylamino)
or electron-withdrawing groups (fluorines) to the addi-
tional aromatic ring or elongating olefinic linkage (Figure
1C). When bound with NG16, these compounds exhibited
significantly increased fluorescence with emission maxima
ranging from 583 to 668 nm (Figure 1D–G), which spans
the spectrum from red to far-red, nearly overlapping the en-
tire existing RFP palette.
A panel of DNA mimics of RFP with different chro-
mophores allows direct comparison of spectral properties
and corresponding substituent’s effect (Table 1). All syn-
thesized chromophores weakly fluoresce in aqueous so-
lution at pH 8 with quantum yields below 0.007, as do
denatured RFP and other reported RFP chromophores
(6,29,31,32), which results from the nonradioactive decay
of their photo-excited state via the intramolecular motion
about the ethylenic bridge (33). The quantum yields of these
RFP chromophores are relatively higher than those of GFP
chromophores partly due to the bulky aromatic group may
hinder the exocyclic C–C bond rotation, and a similar ef-
fect was also observed for the synthetic Kaede chromophore
(32). The observed fluorescence activation by G4 indicated
that G-quadruplex might provide a rigid environment like
the ␤-barrel structure of fluorescent proteins to lock the pla-
nar conformation of the RFP chromophores and prevent
intramolecular motions, which makes fluorescence predom-
inant in energy dissipation of the excited state. This is con-
firmed by the significantly increased excited-state lifetimes
of all G4-chromophores complexes compared to individ-
ual RFP chromophores (Supplementary Figure S4). Un-
der the optimized condition with 100 mM K+ and 50 mM
Mg2+ (Supplementary Figure S5) in the Tris buffer (pH 8.0),
most of the RFP chromophores exhibited at least 50-fold
increase of fluorescence quantum yield (␾) in the presence
of NG16, and the maximum up to 75-fold was observed for
DFHBFSI. The quantum yield of DFHBFSI-NG16 (␭
=
em
606 nm) is above 0.39, which is pronouncedly higher than
that of all reported synthetic RFP mimics, including un-
locked (<0.007) (29,31,32), protein-confined (0.052) (10),
and intramolecularly locked RFP chromophores (0.19) (9).
It is even better than that of most of RFPs with simi-
lar emission maxima (600–620 nm), such as mRuby (0.35)
(5), mCherry (0.22), FusionRed (0.19) (34), and also its
structure parent Kaede (0.33) (35). Substituent compar-
isons would provide useful information for directing the
spectral turning of DNA RFP mimics (Table 1). Replac-
ing the benzene ring (DFHBSI) with a naphthalene ring
(DFHBNI) leads to an 18 nm red shift in excitation and
a 22 nm red shift in emission due to the expanded conju-
gated system. Intriguingly, introducing dimethylamino sub-
stituent (DFHBASI), a strong electron-donating group, on
the benzene ring causes a substantial red-shift in both exci-
tation (35 nm) and emission (30 nm) which is even superior
to the naphthalene-containing chromophore (DFHBNI),
changing the fluorescence from red to far-red. The most re-
markable bathochromic effect derived from olefinic linkage
elongation by an additional double bond, contributing to
an emission maxima change from 620 nm of DFHBASI
to 668 nm of DFHBAPBI. However, DFHBAPBI–NG16
complex have a relatively low quantum yield (0.032) com-
pared to other chromophore-NG16 complexes. Two rea-
sons may account for this phenomenon: (i) the relatively
low quantum yield is conventionally observed for far-red or
near infrared fluorophores, such as the far-red FP eqFP670
(␸ = 0.06) (36) and near-infrared RFP AQ143 (␸ = 0.04)
(37); (ii) its molecular length is too long to perfectly match
the size of G-quartet. As a result, unlike the fixed spectral

Nucleic Acids Research, 2017, Vol. 45, No. 18 10385
creased tissue penetration, reduced out-of-focus photodam-
age, negligible background fluorescence, and high three-
dimensional resolution (42). To the best of our knowledge,
the two-photon properties of synthetic RFP chromophore
analogues are totally unknown. As shown in Figure 2C,
the DFHBMSI–NG16 complex exhibited an intensive two-
photon absorption with a maximum peak at around 750 nm
(Figure 2D), which resulted from unique S0 to Sn electronic
transitions (43). Its TPEF spectrum possesses a maximal
emission peak at 579 nm, which is slightly hypsochromi-
cally shifted relative to its one-photon emission peak (583
nm). Unexpectedly, although DFHBMSI is a RFP chro-
mophore analogue with an unlocked structure, the confin-
ing effect by G4 successfully endow DFHBMSI with de-
cent two-photon properties with a two-photon cross-section
(␴) of 38.6 GM and a two-photon excitation action cross-
section (brightness, ␴ꢁ) of 14.3 GM (Supplementary Table
S5). They are comparable with those of its protein counter-
ꢁ
part, mStrawberry (␴ = 35 GM, ␴ = 12 GM) (43). Com-
Figure 2. (A) Comparison between mCherry and DFHBFSI–NG16 in ex-
citation and emission spectra. (B) Photobleaching curves of DFHBFSI-
NG16 (1 ␮M), Lyso-Tracker Red (1 ␮M) and mCherry (1 ␮M). (C)
Two-photon excitation action cross-section of DFHBMSI (3 ␮M) and
DFHBMSI with NG16 (15 ␮M). (D) Two-photon excited emission spec-
tra of DFHBMSI (3 ␮M) and DFHBMSI with NG16 (15 ␮M, Ex = 750
nm).
pounds DFHBSI and DFHBFSI shows similar two-photon
properties upon G4 binding (Supplementary Table S5). Sig-
nificantly, the S0 to Sn transition of these RFP mimics at
around 750 nm perfectly matches the tuning range of fem-
tosecond Ti:sapphire lasers, which make them well applica-
ble to most of commercial TPEF microscopic systems.
The synthetic RFP chromophore is a selective fluorescent dye
for parallel G4 structures
properties of each RFPs, the emission of DNA RFP mim-
ics can be facilely modulated in a controllable manner by
changing various synthetic chromophores that hold great
potential to be rationally designed.
To our knowledge, the proposed RFP chromophores are
structurally dissimilar to any structural scaffolds of known
G4 ligands or fluorogens, presenting a new category of G4
fluorescent probes. Given that G4s have gained substan-
tial interest due to their increasing biological significance
and applications in biosensing, supramolecular chemistry,
and bionanotechnology (44–46), the development of small-
molecule ligands with fluorogenic ability is highly impor-
tant for G4 structure investigation and G4-targeting drug
discovery (47–51). Thus, the recognition characteristics of
these compounds with G-quadruplexes warrant further ex-
amination. Taking DFHBFSI as a model, the gradual ad-
dition of NG16 to DFHBFSI solution in the presence of
100 mM K+ and 50 mM Mg2+ lead to a distinct red shift
(ꢁ␭ = 25 nm) in absorption with a clear isosbestic point at
534 nm (Figure 3A), indicating that the spectroscopic prop-
erties of DFHBFSI can be altered via a strong interaction
with G4. The fluorescence titration at the same condition
exhibited a 12 nm bathochromic shift of DFHBFSI emis-
sion maxima concomitant with significant fluorescence en-
hancement upon binding with NG16 (Figure 3B). The spec-
tral bathochromic shift of DFHBFSI caused by G4 binding
is probably due to the ␲−␲ stacking interactions between
the phenolic ring of DFHBFSI and the G-tetrad plane (6).
The fluorescent signal of DFHBFSI shows a good linear
response in the range of 0.02–0.5 ␮M of NG 16 with the
lowest detectable concentration of 0.02 ␮M (Supplemen-
tary Figure S7). A 1:1 binding model for DFHBFSI with
NG16 was confirmed by a Job’s plot analysis (Supplemen-
tary Figure S8), and the fitting results of fluorescence titra-
tion curves provided a Kd value of 1.27 ␮M, indicating the
high affinity of DFHBFSI with G4 structure. All the other
Another spectral feature of the new RFP mimics is their
large Stokes shifts. In contrast to small Stokes shifts of con-
ventional RFPs (summarized in Supplementary Table S4),
such as 23 nm for mCherry (shown in Figure 2A), the pro-
posed DNA RFP mimics have large Stokes shifts exceeding
80 nm (shown in Table 1 and Supplementary Figure S6), for
instances 93 nm for DFHBFSI (shown in Figure 2A) and
112 nm for DFHBAPBI. A larger Stokes shift can reduce
the self-quenching and fluorescence detection errors caused
by excitation back scattering effects, which is beneficial for
high contrast fluorescence imaging (38). The large Stokes
shift is probably due to the excited-state relaxation of the
RFP chromophore itself (39). Moreover, the photo-stability
of G4-based RFP mimics was also determined. As depicted
in Figure 2B, the fluorescence signal of DFHBFSI-NG16
complex shows negligible decrease upon continuous illumi-
nation over 30 min, while those of mCherry, a typical RFP,
and Lyso-Tracker Red, a model of small molecular red flu-
orophore, quickly declined 93.5% in 10 min and 80% in 30
min, respectively. This result suggested that the DNA RFP
mimics possess much better photo-stability against photo-
bleaching than RFPs, probably resulting from that partial
exchange of bound chromophore with free chromophore in
solution prevents the accumulation of photo-bleached com-
plexes (24,40,41).
Next, we examined the two-photon fluorescence prop-
erty of the proposed DNA RFP mimics. Two photon
fluorophores, cooperating with two-photon excitation flu-
orescence (TPEF) microscopy, possesses several advan-
tages over conventional one-photon imaging, including in-

10386 Nucleic Acids Research, 2017, Vol. 45, No. 18
Table 1. Photophysical and binding properties of chromophores and chromophore-NG16 complexes
Chromophore
Exmax (nm)
Emmax (nm)
ε (M−1 cm−1
)
Stokes shift (nm)
Q.Y.b
Kd (␮M)
DFHBMSI-NG16
DFHBMSI
DFHBSI-NG16
DFHBSI
DFHBFSI-NG16
DFHBFSI
DFHBNI-NG16
DFHBNI
DFHBASI-NG16
DFHBASI
DFHBAPBI-NG16
DFHBAPBI
mCherry
499
500
503
501
513
511
521
517
538
540
547
548
587
583
570
590
576
606
594
612
600
620
614
668
662
610
16 950
18 200
17 350
19 210
28 200
32 000
37 500
38 840
22 800
23 890
15 500
16 120
72 000
84
70
87
75
93
83
91
83
82
74
121
114
23
0.37
0.0072
0.31
0.0066
0.39
0.0052
0.30
0.0048
0.19
0.0028
0.032
0.0011
0.22
1.56 0.31
N/Aa
1.76 025
N/A
1.27 0.11
N/A
1.37 0.19
N/A
2.77 0.58
N/A
24.88 0.82
N/A
N/A
aNot applicable.
bQuantum yields were calculated using rhodamine 6G (ꢂf = 0.95 in water) as standard for DFHBMSI, DFHBSI, DFHBFSI, DFHBNI and DFHBASI,
and sulfoindo cyanine dye Cy5 (ꢂf = 0.20 in PBS) as standard for DFHBAPBI.
nm and a negative peak at 240 nm (Figure 3C). It marginally
changed upon the addition of DFHBFSI, indicating a slight
stabilizing effect of DFHBFSI on the parallel G4 structure
of NG16 in the presence of K+/Mg2+ salts. It is notewor-
thy that under the condition without metal ions, the par-
allel G4 structure of NG16 is poorly formed, whereas ad-
dition of DFHBFSI significantly improve the parallel G4
formation reflected by a marked enhancement of their CD
characteristic peaks (Figure 3C), indicating its role as the
parallel G4 inducer. Subsequent CD melting experiments
(Figure 3D) showed that DFHBFSI can raise G4 thermal
stabilization as the melting temperature (Tm) of DFHBFSI-
NG16 was 5.4◦C higher than that of NG16 alone in the
cases involving K+/Mg2+ salts, whereas the ꢁTm between
DFHBFSI–NG16 and NG16 further increased to 8.1◦C
without salts. These CD results also evidence the crucial in-
fluence of metal ions on G4 topology and G4-chromophore
interaction (Supplementary Figure S9). This is consistent
Figure 3. (A) Absorption and (B) emission spectra of DFHBFSI (10 and
with the fluorescence optimization data presented above
3 ␮M) upon stepwise addition of NG16. Inset of B is plot of fluorescence
(Supplementary Figure S5). K+ could account for 62% of
enhancement ratio (F/F0) versus the concentration of NG16. (C) Changes
the fluorescence because of its known ability and prefer-
ence to support parallel structures of short loop-sized DNA
G4s. The divalent ion, Mg2+, also enhances fluorescence ac-
tivation in the presence of K+, indicating a possible role
for Mg2+ in improving DFHBFSI binding with negatively-
charged G4 structures via neutralizing its phenolate anion
and screening their electrostatic repulsion (27, 28).
in CD spectra of NG16 (10 ␮M) upon the addition of DFHBFSI (50 ␮M)
in different buffer conditions. K+, 100 mM; Mg2+, 50 mM. (D) CD melting
curves of NG16 (5 ␮M) at 265 nm under different conditions. NG16 (curve
1) and NG16 with DFHBFSI (25 ␮M, curve 2) in buffer without metal
ions; NG16 (curve 3) and NG16 with DFHBFSI (25 ␮M, curve 4) in buffer
containing K+ (100 mM) and Mg2+ (50 mM).
Among the known G4 probes, only a few molecules have
selectivity for G4s over duplex DNA and even fewer are se-
lective for a particular G4 structure (47–51,54,55). To test
its structural specificity, DFHBFSI was challenged by var-
ious DNA conformations (their CD characterizations are
shown in Supplementary Figure S10) to measure its fluo-
rescence response. As depicted in Figure 4A, marked fluo-
rescence enhancement was exclusively observed for parallel
G4s, such as NG16, EAD, and TT3T. The fluorescence in-
crement of DFHBFSI induced by parallel NG16 was ∼35-
fold higher than that by the antiparallel G4 (TBA), 25–
41-fold higher than those by hybrid-type G4s (Hum21, h-
Telo, HT), 42–51-fold higher than those by double-stranded
DNA (Ds26, Ds17), and over 650-fold higher than those
by single stranded DNA (A21 and T21), indicating the
RFP analogues have similarly low Kd value ranging from
1.37 to 2.77 ␮M except for DFHBAPBI (Kd = 24.9 ␮M)
which is consistent with its relative low quantum yield.
Next, we investigated the topology and thermal stabil-
ity of G-quadruplexes upon the addition of DFHBFSI by
using circular dichroism (CD) spectroscopy. G4s are struc-
turally polymorphic and can be classified as parallel, an-
tiparallel, or mixed parallel/antiparallel structures based on
the orientation of the G-tracts in the quartet core (52). The
G4s folding topologies depend on sequences, loop geome-
try, and the local environment, especially the central metal
ions in G4s (53). Under the optimum emission condition
(25 mM Tris-HCl buffer, 100 mM KCl, 50 mM MgCl2 and
pH 8.0), the CD spectra of NG16 exhibited a typical char-
acteristic of a parallel structure with a positive peak at 265

Nucleic Acids Research, 2017, Vol. 45, No. 18 10387
Figure 4. (A) Fluorescence changes of DFHBFSI (3 ␮M) in response to
G-quadruplex (15 ␮M), double-strand (15 ␮M) and single-strand oligonu-
cleotides (15 ␮M). F and F0 are the fluorescence intensity of DFHBFSI in
the presence and absence of the oligonucleotides, respectively. (B) Fluo-
rescence responses of G-quadruplex-specific dyes (3 ␮M) toward different
kinds of oligonucleotides (15 ␮M). (C) Reversible topological switch be-
tween parallel and anti-parallel G-quadruplexes. (D) Real-time monitor-
ing the topological switch of G4 based on DFHBFSI-PW17 (1 ␮M). K+,
2 mM; Mg2+, 50 mM; Pb2+, 150 ␮M; EDTA, 150 ␮M.
Figure 5. Radiationless decay of RFP chromophore (DFHBFSI) via
twisted intramolecular charge-transfer state. (A) Equilibrium geometry
configurations at the minimum energy point of ground state S0 (S0-min)
and the minimum energy point of excited state S1 (S1-min). (B) Poten-
tial energy surfaces of S1 (red) and S0 (blue) as well as oscillator strength
(fosc) along the phenoxy-twisted photoisomerization pathway. (C) Charge
density difference isosurfaces (isovalue = 0.0075) at the phenoxy-twisted
minimum energy conical intersection (MECI) between S1 and S0 (S0/S1
MECI, ␸ = 55o). Positive isosurfaces are red, and negative isosurfaces are
blue.
RFP chromophore is a topology-specific turn-on probe for
parallel G4s. In a sharp contrast, several widely used G4-
binding dyes, includes thiazole orange (TO) (56), crystal vi-
olet (CV) (57), and N-methyl mesoporphyrin IX (NMM)
(58), show poor discriminating abilities for parallel G4s
from other G4 or DNA conformations (Figure 4B). Mean-
while, native gel electrophoresis experiments also confirmed
that DFHBFSI has a good specificity for parallel G4 struc-
tures (Supplementary Figure S11), demonstrating the po-
tential of DFHBFSI as a gel-staining reagent for visualizing
parallel G4s. A dose–response experiment showed that G-
quadruplex (NG16) is still detectable at 0.25 ␮M, indicating
an approximate detection limit of 25.6 ng for gel staining.
The unique specificity of DFHBFSI for parallel G4 struc-
ture enables dynamic monitoring of topological switching
of G4s in real-time, which is seldom explored in previous re-
search on G4-targeting dyes. Due to the higher efficiency of
Pb2+ in stabilizing G4 structures, substituting K+ by Pb2+ as
the G4 chelating cation causes a topological transformation
from parallel to anti-parallel G4 (59), presenting an ideal
model of G4 structural switch. As depicted in Figure 4C and
D, in the presence of DFHBFSI, Pb2+-driven parallel-to-
antiparallel conformation transition can be directly moni-
tored by sharp quenching of fluorescence at 606 nm to 14%
as a result of negligible fluorogenic response of DFHBFSI
to antiparallel G4s. Introducing a Pb2+ chelator, EDTA,
quickly restored fluorescence to 95%, indicating that the ex-
traction of Pb2+ by EDTA from Pb2+-G4 complex leads to
re-formation of parallel G4s via re-binding with K+. Fur-
ther CD measurements confirmed this K+/Pb2+-driven G4
conformational switch (Supplementary Figure S12). This
clearly demonstrated that the RFP chromophore is a smart
G4 dye competent to visualize G4 structural switches in
real-time, which is highly promising for functional investi-
gation of G4 and G4-based DNA nano-machines.
Theoretic calculations and molecular dynamics simulations
for elucidating fluorogenic G4 RFP mimics
To gain more insight into the mechanism of the on/off op-
tical switch of RFP chromophores upon binding with G4,
long-range-corrected time-dependent density functional
theory (LRC-TD-DFT) calculations and molecular dynam-
ics (MD) simulations were performed using DFHBFSI as a
model. In the case of anionic form of DFHBFSI which is
dominant in neutral pH, LRC-TD-DFT calculation results
shows that the minimum energy conformation in the first
excited state (S1-min) is substantially non-planar with an
almost perpendicular plane of the phenolic ring, whereas its
counterpart in the ground state (S0-min) is nearly coplanar
(Figure 5A). Further coordinate-driving potential surface
scans indicate that the potential energy surfaces of S0 and
S1 are approaching upon increasing the twist angle (␸) at
the bridging bond of the phenolic moiety. When DFHBFSI
in its S0-min state is excited to S1 by photoirradiation, the
relaxation process starts with the barrierless rotation of the
phenolic moiety to ␸ = 55◦, leading to the minimum energy

10388 Nucleic Acids Research, 2017, Vol. 45, No. 18
by the competitive inhibition of DFHBFSI-NG16 fluo-
rescence by 5, 10, 15, 20-tetra (N-methyl-4-pyridyl) por-
phyrin (TMPyP4) (Supplementary Figure S13), a known
end-stacking ligand of G4 (62). With respect to intramolec-
ular motion of DFHBFSI (Figure 6B), in its free form, the
phenyl moiety was constantly rotating (50% of the simula-
tion), which broke the conjugated system essential for high
quantum yield therefore it does not fluoresce. In contrast,
the phenyl moiety of DFHBFSI complexed with NG16
rarely rotated (∼0.01% of the simulation), evidencing that
the strong ␲-␲ stacking interaction between DFHBFSI and
G-quartet significantly restricts the chromophore’s range of
motion and improve its rigidity. NG16-confined DFHBFSI
exhibited a stable conformer with 14◦ and 12◦ for the di-
hedral angles of phenoxy-bridge and imidazolinone-bridge
bond torsion, respectively (Figure 6A). Such torsion angles
are very close to planarity and comparable to those of the
mCherry chromophore (13.7◦ and 11.3◦) (3), which leads to
increased fluorescence.
Furthermore, MD simulations are also helpful for elu-
cidating the G4 topological preference of DFHBFSI. A
hybrid-type NMR G-quadruplex structure for HT (PDB
ID: 2GKU) (63) and an antiparallel NMR G-quadruplex
structure for TBA (PDB ID: 5MJX) (64) were also used.
The examined models and the estimated free energies of the
binding of DFHBFSI with NG16, HT, and TBA are shown
in Figure 6. Unlike the perfect stacking of DFHBFSI on
the ending G-quartet of NG16 which confined DFHBFSI
to the co-planar conformation, DFHBFSI bound to both
HT and TBA twisted dramatically to form favorable inter-
actions with both G-quartet and several bases in the flank-
ing and loop sequences, for instances T13, A14, A24 in
HT and G8 in TBA, respectively (Figure 6C and D). As
a consequence, both the conformations of DFHBFSI in
their complexes with HT and TBA are considerably non-
planar and thus adverse to its fluorescence. In addition,
DFHBFSI exhibited stronger binding with NG16 (–27.6
kcal/mol) than with HT (–11.3 kcal/mol) and TBA (–12.7
kcal/mol), which show good agreement with the observed
G4 selectivity of DFHBFSI. Overall, the quantum chem-
ical calculation and molecular modeling reasoned the flu-
orogenic property of DFHBFSI upon G4 binding and its
unique specificity to parallel G4.
Figure 6. Averaged structures of DFHBFSI and G-quadruplex-
DFHBFSI obtained through MD simulations. (A) Complex model of
DFHBFSI with NG16. (B) The flipping of phenoxy ring in DFHBFSI in
100 ns MD simulation. In the y axis, 0 stands for no flipping, and 1 stands
for flipping. Flipping is defined as the phenoxy ring rotates across the
plane perpendicular to the imidazole ring. Complex models of DFHBFSI
with (C) HT and (D) TBA. DFHBFSI, G-quartet, adenine and thymine
are shown using green, yellow, red and blue sticks, respectively.
conical intersection (MECI) between S1 and S0 (Figure 5B).
Electron density analysis suggest that in this highly twisted
S1 state, the electronic charge obviously transfers from phe-
nolic moiety to imidazolinone, vinyl linkage, and aromatic
ring moieties (Figure 5C). This intersection causes a twisted
intramolecular charge transfer (TICT) to facilitate radia-
tionless relaxation, and results in an extremely low oscillator
strength (f1) of 0.0005 (Figure 5B), implying a low fluores-
cence. This deactivation pathway is similar to that reported
previously for the green and red fluorescent protein chro-
mophores (33,60). Hence, the LRC-TD-DFT results mani-
fested that the proposed RFP chromophores without con-
finement by G4 are non-fluorescent due to intramolecular
rotation-caused TICT.
In order to further elucidate G4-activable DFHBFSI flu-
orescence, MD simulations were carried out to investigate
the binding mode and interaction of G4 and DFHBFSI.
A parallel NMR G-quadruplex structure for NG16 (PDB
ID: 2LXV) (61) was used as a template. The starting con-
formations of the DFHBFSI-NG16 complex were obtained
by molecular docking using Autodock, and then the MD
simulations were performed using GROMACS for 100 ns
molecular dynamic runs. Three representative complexes
as starting conformations were simulated: (i) DFHBFSI
with end stacking mode; (ii) DFHBFSI with groove bind-
ing; (iii) free DFHBFSI as a control. The simulation re-
sults showed that the groove-binding complexes were con-
siderably unstable, in which the DFHBFSI quickly moved
away from the G4 after roughly 10 ns (data not shown),
whereas DFHBFSI perfectly end-stacks on the NG16 struc-
ture throughout simulation (Figure 6A). The end-stacking
mode of DFHBFSI was further experimentally supported
One- and two-photon bio-imaging in live cells and tissues
Encouraged by their prominent spectral merits, includ-
ing bright red luminescence, large Stokes shifts, anti-
photobleaching and two-photon fluorescent properties, we
further exploited DNA mimics of RFP as fluorescent
probes for living cell and tissue bio-imaging. DFHBFSI
was chosen as the model because it had the highest
quantum yield of all the proposed RFP chromophores.
Prior to imaging experiments, we confirmed that the
G4-activated fluorescence of DFHBFSI was barely in-
fluenced by the presence of cell lysates and cell culture
medium with or without live cells (Supplementary Figure
S14). The cell counting kit-8 (CCK-8) assay revealed that
DFHBFSI has negligible influence on cell viability over
the 24-h observation period (Supplementary Figure S15).
These results imply the reliability and biocompatibility of

Nucleic Acids Research, 2017, Vol. 45, No. 18 10389
Figure 8. (A) Time-lapse confocal microscope images of PTK RMFP
treated CCRF-CEM cells with transfection of pDispaly-GFP for differ-
ent time spans. The images were acquired using 488 nm excitation and
presented as overlay images (top) from 500 to 550 nm emission channel
(green) and 570–620 nm emission channel (red), respectively, Scale bar:
5 ␮m. Co-localization analysis of red and green fluorescence channels of
upper images (bottom). (B) Long duration confocal microscope image at
different time span of PTK RMFP treated CCRF-CEM cells at 570–620
nm emission channel using 488 nm excitation, Scale bar: 5 ␮m.
Figure 7. (A) Confocal microscope image of PTK RMFP treated CCRF-
CEM cells and Ramos cells at 570–620 nm emission channel using 488 nm
excitation, Scale bar: 20 ␮m. (B) Flow cytometry of PTK RMFP treated
CCRF-CEM cells and Ramos cells. (C) Confocal microscope image of
PTK RMFP treated Ramos cells with transfection of pcDNA3-PTK7 at
570–620 nm channel, Scale bar: 20 ␮m. (D) Relative mRNA level of PTK7
in Ramos cells with transfection of pcDNA3-PTK7 using real-time quan-
titative reverse transcription PCR. Values are means with SD (n = 3), **P
< 0.001.
DFHBFSI-G4 complexes for bio-imaging. To selectively
target protein biomarkers displaying on cancer cell sur-
face, DFHBFSI-G4 complexes was employed as RFP mim-
ics to specifically ‘tag-and-lighten’ cancer-associated mem-
brane proteins through conjugation with their targeting-
aptamers. Aptamers are single-stranded DNA or RNA
oligonucleotides with unique molecular recognition and
binding capabilities specific to various targets, including
small molecules, proteins, and live cells (65–67). The pro-
posed membrane protein-specific RFP-mimicking fluores-
cent probe (RMFP) is composed of two segments: a re-
porter strand bearing two repeat NG16 sequences binding
with DFHBFSI and a recognition strand with the aptamer
sgc8 targeting to protein tyrosine kinase-7 (PTK7), a trans-
membrane receptor protein closely relative to a number of
cancers (68–71), which was readily prepared by simple hy-
bridization (Supplementary Table S6). As shown in Figure
7A, based on highly specific aptamer recognition, PTK7
RMFP are selectively targeted to CCRF-CEM cells, a T
cell acute lymphoblastic leukemia (ALL) cell line with high
expression of PTK7, reflected by bright red fluorescence
Figure 9. (A) Two-photon image of PTK RMFP treated CCRF-CEM cells
at using 750 nm excitation, Scale bar: 20 ␮m. (B) 3D reconstruction from
two-photon images of PTK RMFP treated tissue section from CCRF-
CEM cells xenograft using 750 nm excitation, Scale bar: 100 ␮m.

10390 Nucleic Acids Research, 2017, Vol. 45, No. 18
on the cellular periphery, whereas the control Ramos cells
shows weak fluorescence. It is noteworthy that DFHBFSI,
by itself, is not detectably fluorescent upon incubation with
either CCRF-CEM cells or Ramos cells, indicating negli-
gible background. Flow cytometry results (Figure 7C) also
revealed that the PTK7-specific RMFP selectively binds to
CCRF-CEM cells. To further elucidate the specificity of
PTK7-targeting RMFP, we engineered Ramos cells by elec-
troporation with plasmids expressing PTK7. Fluorescence
was readily detectable on the cytoplasmic membrane of in-
dividual engineered Ramos cells (Figure 7D), which accords
with the quantification data of PTK7 mRNA by real-time
quantitative reverse transcription PCR (Figure 7E). These
data suggested that the proposed RMFP is a feasible tool
for probing expression profiles of PTK7 in different cells.
To explore the plasma membrane localization of PTK7
RMFP, we transfected CCRF-CEM cells with pDisplay-
EGFP for a co-localization analysis. The pDisplay sys-
tem is a mammalian expression vector that allows display
of enhanced GFPs (EGFPs) on the extracellular side of
cell plasma membrane. Given that the large Stokes shifts
of DFHBFSI-G4 complexes (Ex = 513 nm, Em = 606
nm) greatly facilitates simultaneous imaging of multi-color
fluorophores, RMFP and EGFP were simultaneously ex-
cited by the same 488 nm channel and monitored sepa-
rately by emission channels of 500–550 nm and 570–620
nm, respectively, without mutual interference. A perfect co-
localization of RMFP and EGFP was observed (Figure
8A) and further quantified using Manders’ overlap coeffi-
cient (R). A high overlap coefficient (0.90) was obtained and
confirmed that PTK7-bound RMFP was predominately lo-
cated on the membrane extracellular surface. Moreover,
this RMFP/EGFP system is desirable for a simultaneously
comparison of the anti-photo-bleaching properties of flu-
orescent protein and its DNA mimics. As shown in Fig-
ure 8A, DFHBFSI–G4 complexes retain distinctive red
fluorescence throughout 30 min irradiation. In contrast,
EGFP quickly lost its fluorescence in 10 min irradiation
due to severe photo-bleaching. Furthermore, the real-time
imaging of a single live CCRF-CEM cell confirmed strong
membrane staining could be still measured even after 2 h
continuous observation, which allows monitoring the dy-
namic processes of membrane proteins, such as cellular in-
ternalization of PTK7 (Figure 8B). These results proved
that DNA mimics of RFPs are suitable for long-term and
real-time cellular imaging due to their superior anti-photo-
bleaching properties.
specificity of aptamer-derived RMFP probes in living cells
or tissues. As a proof-of-concept, PTK7 RMFP-treated
CCRF-CEM cells were employed for two-photon imaging
upon excitation of the sample at ␭ = 750 nm. As shown
in Figure 9A, strong red fluorescence was detected on the
surface of CCRF-CEM cells. Similar two-photon imaging
was further undertaken by incubating the PTK7 RMFP
probe with tissue sections from CCRF-CEM cell xenograft
mice. The images were collected at different depths and
reconstructed in a three-dimensional box (3D) to present
the spatial distribution of PTK7-overexpressed cells (Fig-
ure 9B), and the fluorescence of RMFPs could be evidently
detected at depths of ∼80 ␮m. The bright fluorescence de-
tected clearly indicated that the developed probe could effi-
ciently monitor PTK7 expression in real tissue samples.
CONCLUSIONS
In summary, we have successfully developed a palette of
G4-chromophore complexes spanning most of spectrum re-
gion of existing red fluorescent proteins, providing an un-
precedented complement to conventional synthetic mimics
of GFPs. These novel RFP chromophore analogues, which
possess an extended ␲-ring system, not only cause a signif-
icant emission shift to red and far-red but also strengthen
the interactions with DNA G4 structures via ␲-␲ stack-
ing, presenting a simple, potent, and versatile strategy to
develop highly emissive RFP mimics via G4 binding and
activation. Detailed theoretical calculation and molecular
dynamics simulations suggest that the confinement effect
of G4 is analogous to RFP, in which the end-stacking
mode of RFP chromophores bound to G4 greatly restricts
their intramolecular rotation and TICT-induced radiation-
less relaxation, ensuring their bright fluorescence. These
DNA RFP mimics offer attractive photo-physical advan-
tages over RFPs in that: spectral properties are readily tun-
able by changing specific synthetic chromophores; large
Stokes shifts enable high-contrast multicolor imaging us-
ing only a single excitation; and excellent resistance to pho-
tobleaching allows long-term real-time monitoring of live
cells. Furthermore, despite the unlocked structure of RFP
chromophores, G4 confinement endows the RFP mimics
with a unique S0 to Sn two-photon excitation with red emis-
sion, which is highly desirable for in vivo imaging. On the
other hand, the proposed RFP chromophores affords a
novel and distinctive category of G-quadruplex probes, pos-
sessing a selective light-up function specific for parallel con-
formation, which is greatly useful for discrimination of dif-
ferent G4 topologies and real-time detection of G4 struc-
tural switches. This work will shed light on multifaceted re-
searches: (i) the discovery of a new generation of FP mim-
icking system with valuable features, including red, near-
infrared, and two-photon fluorescence; (ii) the development
of novel topology-selective G4 probes for G4-relative detec-
tion and nano-system; (iii) potential toolkits for biomedical
imaging and clinical diagnosis based on the integration of
DNA RFP mimics with functional nucleic acids.
The generality of DNA RFP mimics as potent probes for
tagging cell membrane biomarkers was also tested. We fur-
ther prepared RMFPs targeting to hepatocyte growth fac-
tor receptor (HGFR) or epithelial cell adhesion molecule
(EpCAM), two representatively overexpressed tumor anti-
gens, through integrating with their specific aptamers, SL1
(72) and SYL3C (73), respectively. Both RMFPs exhibit
great potency for cell-specific imaging based upon target-
expression differences (Supplementary Figures S16 and
S17), which can efficiently discriminate targeting antigen-
positive cells from negative cells.
Taking advantage of the two-photon properties of
DFHBFSI/G4 complexes, we further investigated the pos-
sibility of conducting two-photon imaging based on the
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.

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NSF of China [21235002, 21575038]; Foundation for In-
novative Research Groups [21521063]; Young Top-notch
Talent for Ten Thousand Talent Program; NSF of Hunan
Province [2015JJ1005]. Funding for open access charge: Na-
tional Natural Science Foundation of China.
Conflict of interest statement. None declared.
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