Pursuing origins of (poly)ethylene glycol-induced G-quadruplex structural modulations
Nucleic Acids Research
Pursuing origins of (poly)ethylene glycol-induced G-quadruplex structural modulations
Marko Trajkovski 2
Tamaki Endoh 1
Hisae Tateishi-Karimata 1
Tatsuya Ohyama 1
Shigenori Tanaka 0
Janez Plavec 2 4
Naoki Sugimoto sugimoto@konan- 1 3
0 Department of Computational Science, Graduate School of System Informatics, Kobe University , 1-1, Rokkodai, Nada-ku, Kobe 657-8501 , Japan
1 Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University , 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047 , Japan
2 Slovenian NMR Centre, National Institute of Chemistry , Hajdrihova 19, Ljubljana, SI-1000 , Slovenia
3 Graduate School of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University , 7-1-20 Minatojima-Minamimachi, Kobe 650-0047 , Japan
4 Faculty of Chemistry and Chemical Technology, University of Ljubljana , Vecˇna pot 113, p. p. 537, SI-1000 Ljubljana , Slovenia
Molecular crowding conditions provided by high concentration of cosolutes are utilized for characterization of biomolecules in cell-mimicking environment and development of drug-delivery systems. In this context, (poly)ethylene glycols are often used for studying non-canonical DNA structures termed Gquadruplexes, which came into focus by emerging structural biology findings and new therapeutic drug design approaches. Recently, several reports were made arguing against using (poly)ethylene glycols in role of molecular crowding agents due to their direct impact on DNA G-quadruplex stability and topology. However, the available data on structural details underlying DNA interaction is very scarce and thus limits in-depth comprehension. Herein, structural and thermodynamic analyses were strategically combined to assess G-quadruplex-cosolute interactions and address previously reported variances regarding the driving forces of G-rich DNA structural transformations under molecular crowding conditions. With the use of complementary (CD, NMR and UV) spectroscopic methods and model approach we characterized DNA G-quadruplex in the presence of the smallest and one of the largest typically used (poly)ethylene glycols. Dehydration effect is the key contributor to ethylene-glycol-induced increased stability of the G-quadruplex, which is in the case of the large cosolute mainly guided by the subtle direct interactions between PEG 8000 and the outer G-quartet regions.
In cells, the genome is encoded in DNA, which canonically
forms duplex through Watson-Crick base pairing. When
the genetic information is replicated or expressed into
functional proteins, the DNA duplex is temporarily dissociated
by proteins such as DNA polymerase, RNA polymerase
and helicase allowing the dissociated single stranded DNA
to form non-canonical structures (
). One of the stable
non-canonical structures that have been demonstrated to
exist in living cells recently are G-quadruplexes (
are formed by G-rich sequences of both DNA and RNA.
Various biological functions of the DNA and RNA
Gquadruplexes that regulate gene expression processes such
as replication, transcription, post-transcriptional RNA
processing and translation have been demonstrated by
biological and biochemical functional studies (
The basic building block of a G-quadruplex is G-quartet,
which consists of four guanine nucleobases circularly
interacting through Hoogsteen-type hydrogen-bonds (
). Pile of
stacked G-quartets represents the core of a G-quadruplex
structure. Although with some exceptions in
intramolecular G-quadruplexes, the guanines located in the same
corner of the stacked G-quartets are generally consistent with
respect to the primary sequence and make backbone
pillars like corner posts. While hitherto all the known RNA
G-quadruplexes are monomorphic in that the strands in the
core of the structure are oriented uniformly in the same
direction and linked by three propeller-type loops, extreme
polymorphism is the hallmark of G-rich DNA. Moreover,
the variety of folding topologies accounts for several
possible loop orientations, which are related to different
orientations of the four stems in the G-quadruplex core and to
alternative glycosidic bond arrangements, i.e. syn or anti
conformation of 2 -deoxyguanosines comprising G-quartets
). Besides propeller type loops found in parallel topology
), the diagonal and lateral loop orientations are
characteristic for anti-parallel and hybrid topologies (
Notorious polymorphism of DNA G-quadruplex topologies
is expanded further by peculiar loop orientations such as
V-loop, D-loop and bulges (
). Additionally, DNA
Gquadruplex topologies depend not only on the length and
sequence of the loops, but also on the molecular
environment such as nature and concentration of cations in
Intracellular molecular environment is characterized as
highly concentrated solution of various cosolutes, most
notably proteins, nucleic acids and metabolites, which
make up the so called molecular crowding conditions (
The molecular crowding condition has been demonstrated
to stabilize the G-quadruplex structures, while
destabilize canonical duplexes (
). Additionally, it was shown
that the equilibrium of different G-quadruplex structures
adopted by G-rich DNA from human telomeric region was
severely perturbed by supplementing aqueous solution with
polyethylene glycol (PEG), which triggered conversion of
hybrid and antiparallel topologies into parallel-stranded
). These observations suggested that the
parallel G-quadruplexes are most relevant in biological
context, thereby establishing the need for further detailed
investigations of their structures and physicochemical
properties under conditions mimicking cellular environment.
However, the polymorphic nature of DNA G-quadruplex
structures and conformational transition depending on
solution conditions and even during thermodynamic
analyses make it difficult to perform such analyses in a
quantitative manner. Interpretation of the experimental results is
further complicated by the intricate mechanisms by which
the commonly used crowding agents such as PEG
influence DNA structural properties. In this regard, DNA
features in the presence of PEG that had been discussed in
terms of excluded volume and lowered water activity effects
were challenged recently by ascribing PEG-induced DNA
structural transformations to preferential and direct
binding of the PEG to G-quartets of parallel-stranded DNA
). Additionally, insights into peculiarity of
PEG and G-quadruplex interactions were expanded by
exploring the interplay of metal ion- and cosolute-depended
structural features (
), which further substantiated direct
binding of PEG to parallel-stranded G-quadruplexes.
Evidently, the interaction modes and moreover structural
impacts of PEG on DNA G-quadruplexes need to be
High-resolution NMR spectroscopy is a powerful
technique for investigating molecular structure and
dynamics with atomic resolution. On this account
we assessed solution-state NMR data on naturally
occurring G-rich DNA oligonucleotide and its
derivatives, including artificially designed sequences in search
for the model amenable for high-resolution studies.
Among the screened DNA oligonucleotides, M2, 5
d[TAGGGACGGGCGGGCAGGGT]-3 , was
particularly interesting due to its distinguished characteristic to
adopt a well-defined G-quadruplex structure in dilute
solution and at different molecular crowding conditions.
In addition, the primary sequence of M2 is consistent
with the vastly accepted G-quadruplex consensus motif
of four GGG tracts intervened by non-G residues. In this
study, structural and thermodynamic analyses of M2 were
strategically combined to assess G-quadruplex-cosolute
interactions. Two grossly different cosolutes, ethylene glycol
(EG) and its polymeric derivative polyethylene glycol 8000
(PEG 8000) were selected to address previously reported
variances of the crowding agents’ effects. EG corresponds
to the basic building block of PEGs, such as PEG 8000,
which is one of the largest typically used crowding agents.
MATERIALS AND METHODS
DNA oligonucleotides were synthesized on H-8 synthesizer
(K&A LaborGera¨te) with the use of standard
phosphoramidite chemistry and deprotected with aqueous
ammonia. Purification and desalting of DNA oligonucleotide was
done with the use of RP-HPLC and Amicon-15 centrifuge
filter with the cutoff 3000. Samples for NMR measurements
were prepared in 100 mM aqueous KCl and 20 mM
potassium phosphate, while the concentrations of EG and PEG
8000 were in the range between 10 and 20 wt%. The
temperature treatment of samples included 5 min incubation at 90–
95◦C immediately followed by cooling in an ice-water bath
for 5 min. For CD and UV analyses, DNA oligonucleotide
was purchased from Japan Bio Services Co., Ltd.
NMR data were collected on Varian NMR Systems
operating at 600 and 800 MHz in temperature range between
5 and 35◦C with most of the experiments being performed
at 25◦C. 1H NMR samples were prepared in 90%/10%
H2O/2H2O or 100% 2H2O at oligonucleotide
concentrations between 0.1 and 1.0 mM per strand. NOESY spectra
were acquired at mixing times between 80 and 300 ms with
the use of DPFGSE solvent suppression method.
DQFCOSY spectra and TOCSY spectra with mixing time of
80 ms were acquired in 100% 2H2O. Twenty different
gradient strengths (2.4–59.6 G cm−1) were used in diffusion
NMR experiments (DOSY). NMR spectra were processed
and analysed with the use of VNMRJ (Varian Inc.) software
and Sparky (UCSF) software (
Restraints and structure calculations
NOESY spectra with mixing times of 80, 150 and 200 ms
were recorded to collect distance restraints for
exchangeable and non-exchangeable protons. The NOE cross-peaks
were classified as strong (1.8–3.6 A˚), medium (2.6–5.0 A˚ )
and weak (3.5–6.5 A˚) relative to the distance-restraint
reference of cytosine H5-H6 protons corresponding to 2.5 A˚.
Glycosidic torsion angle for guanine, thymine and
cytosine residues were restrained to 240 ± 70◦. South-type
sugar puckering for all residues was inferred from large
3JH1 -H2 coupling constants measured in DQF-COSY
spectra. Correspondingly, conformational space related to
pseudorotation phase angles was constraint in the range from
C3 -exo to C1 -exo canonical forms by defining the
endocyclic torsion angles 1 (30 ± 7.5◦), 2 (-30 ± 10◦), 3 (10
± 20◦) and 4 (5 ± 25◦). AMBER 14 software (
parmbsc0 force field with parm OL4 (31) and parm ε/
) modifications and generalized Born implicit
solvation model were used in calculations. For each simulated
annealing calculation random velocity, collision frequency
of 5 ps−1 and the cutoff for non-bonded interactions of
5000 A˚ were used. Twenty structures out of the total 100
were selected based on the smallest energy and amongst
them 10 with the lowest NOE violations were subjected to
a maximum of 10 000 steps of steepest descent energy
minimization. The details of the SA protocols are given in the
Supplementary Data. The resulting families of 10 structures
at the three different conditions were used for structural
statistics, which are for the M2 G-quadruplexes formed in
the absence of cosolutes, in the presence of 20 wt% EG and
10 wt% PEG 8000 reported in Tables 2, 3 and 4,
respectively. UCSF Chimera software (
) was used for
visualization of the calculated structures and preparation of Figures
5, 8 and Supplementary Figures S6-S9. Coordinates of the
M2 G-quadruplex structures in the absence of cosolutes, in
the presence of 20 wt% EG and 10 wt% PEG 8000 have been
deposited in the Protein Data Bank with accession codes
5NYS, 5NYT and 5NYU, respectively.
DNA oligonucleotide (10 M) was dissolved in buffer
containing 20 mM potassium phosphate (pH 7.0), 100 mM KCl
in the absence of cosolutes and in the presence of 20 wt%
EG and PEG 8000, and refolded from 95 to 25◦C at 0.5◦C
min−1. CD spectra of the samples from 200 to 350 nm were
collected on a JASCO J-1500 spectropolarimeter at 25◦C in
1.0-mm path length cuvettes. To measure temperature
dependent CD spectra, DNA oligonucleotide (10 M) was
refolded from 95 to 10◦C at 0.2◦C min−1 in a buffer
containing 20 mM potassium phosphate (pH 7.0) in the
absence of cosolutes and in the presence of 20 wt% EG, 10
wt% PEG 8000 and 20 wt% PEG 8000. CD spectra of the
samples from 200 to 350 nm were collected from 10 to 95◦C
at every 5◦C interval in 1.0-mm path length cuvettes.
DNA oligonucleotide (10 M) was refolded from 95 to
10◦C at 0.5◦C min−1 in buffer containing 20 mM
potassium phosphate (pH 7.0) in the absence of cosolutes and in
the presence of 20 wt% EG, 10 wt% PEG 8000 and 20 wt%
PEG 8000. UV absorbance at 295 nm was measured from
25 to 95◦C at 0.2◦C min−1. To evaluate stability of the
Gquadruplex depending on potassium ion concentration, UV
absorbance of DNA oligonucleotide (5 M) was measured
at 295 nm in a buffer containing 50 mM MES–LiOH (pH
7.0) and in the absence of cosolutes and in the presence of
20 wt% EG and PEG 8000. The thermodynamic stabilities
at 25◦C ( G◦25) were calculated from the fit of the melting
curves to a theoretical equation for an intramolecular
association as described previously (
Measurements of physical properties of solutions
The water activity was determined by the osmotic stressing
method via vapor phase osmometry using a model 5520XR
pressure osmometer (Wescor, Logan, UT, USA) at 25◦C.
The dielectric constants were obtained from the maximum
emission wavelength ( max) of the fluorescence of
8-anilino1-naphthalenesulfonic acid (ANS) at 25◦C excited by 380
). The standard curve was determined by max of
ANS with different contents of ethanol. The dielectric
constants of ethanol standard solutions were measured by an
M-870 liquid dielectric constant meter (Scientifica).
M2 adopts the same G-quadruplex topology in the absence and in the presence of EG and PEG 8000
CD spectra of M2 were analyzed under diluted solution
conditions and compared with data obtained under
molecular crowding conditions induced by 20 wt% EG and PEG
8000 as cosolutes (Figure 1). All CD spectra show clear
positive peaks around 208 and 265 nm and a negative peak
around 243 nm, which are consistent with M2 adopting a
parallel G-quadruplex irrespective of the additional
Imino proton signals are not observable in 1H NMR
spectrum of the desalted sample of M2 which suggests
absence of a well-defined high order structure (data not
shown). Upon addition of potassium ions twelve imino
signals are observed in the range between 11.07 and 11.95
ppm corresponding to twelve guanine residues assembled
via Hoogsteen hydrogen-bonding within one predominant
G-quadruplex structure (Figure 2A). Assignment of the
imino as well as the aromatic, methyl and most anomeric
protons was accomplished with the use of NOESY (Figure
3A), JRHMBC (Supplementary Figure S1) and TOCSY
2D NMR experiments (chemical shifts are reported in
Supplementary Table S1). The NOE-derived correlations
between the imino H1 and aromatic H8 protons (Figure 4A)
are consistent with the following hydrogen-bond
directionalities within the three G-quartets: G3→G8→G12→G17,
G4→G9→G13→G18 and G5→G10→G14→G19
(Figure 5B). In addition, intra- and inter-G-quartet
iminoimino NOE interactions are consistent with M2
adopting a unimolecular G-quadruplex structure exhibiting three
consecutively stacked G-quartets with all four strands in
parallel orientation (Supplementary Figure S2A).
Furthermore, the characteristic interresidual H8-H1 , H8-H2 /H2
as well as sequential guanine(i)-guanine(i+1) NOE
crosspeaks reveal that all the guanine residues are in anti
glycosidic conformation. Notably, the intramolecular M2
Gquadruplex fold is predominant species at up to 1 mM DNA
oligonucleotide concentration (Supplementary Figure S3).
Well-resolved resonances are observed in 1H NMR
spectra of M2 in buffer solution containing potassium ions as
well as in the presence of 20 wt% EG and 20 wt% PEG 8000
(Figure 2B and C). Most important regarding the focus of
this study is observation of twelve major 1H NMR signals
in the imino region characteristic of Hoogsteen
hydrogenbonded guanine residues, which is consistent with
formation of a single G-quadruplex species exhibiting three
Gquartets under all three conditions. Noteworthy, 1H NMR
spectra of M2 without added cosolutes and at 20 wt% EG
are characterized by narrow signals, while broadening of the
1H NMR signals in the presence of 20 wt% PEG 8000
implies equilibrium of different species or slowed down
molecular tumbling. Moreover, DNA concentration of 0.2 mM,
which is much higher in comparison to the one used in
CD spectrum analysis (0.01 mM), might in addition to
formation of predominant G-quadruplex lead to high-order
species and aggregation. In this regard, further NMR-based
characterization of M2 at 10 wt% PEG 8000 gave rise to
narrow and well-resolved 1H NMR signals (Figure 2D).
2D NMR experiments were utilized in assigning of all
imino, aromatic, methyl and most anomeric 1H NMR
chemical shifts corresponding to M2 at 20 wt% EG and
10 wt% PEG 8000 (chemical shifts are reported in
Supplementary Tables S2 and S3, respectively). Detailed
examination of intra- and interresidual NOE cross-peaks
demonstrated that M2 adopts a unimolecular G-quadruplex
comprising three successively stacked G-quartets and
exhibiting all strands in a parallel orientation in the presence of
either of the two cosolutes (Figures 3B–D, 4B–D and 5).
Further analysis of NOE interactions, in particular
intraresidual aromatic-anomeric contacts showed that all guanines
exhibit anti conformation of glycosidic torsion angle.
InterG-quartet imino-imino as well as imino-H8 proton NOE
interactions established formation of G3–G8–G12–G17, G4–
G9–G13–G18 and G5–G10–G14–G19 quartets
(Supplementary Figure S2b-d). Altogether, NMR data collected for
M2 at 20 wt% EG, 10 wt% PEG 8000 and 20 wt% PEG
8000 demonstrate prevalence of a parallel G-quadruplex
topology under molecular crowding conditions as well as
in potassium ion containing solution.
Increase in thermodynamic stability of the M2 G-quadruplex induced by EG and PEG 8000 is coupled with higher potassium ion uptake
Thermodynamic stability of the M2 G-quadruplex was
evaluated by using UV melting analysis. Since the M2
Gquadruplex does not melt even at 90◦C in the presence of
100 mM KCl and cosolutes (data not shown), the melting
properties were evaluated in a basal buffer containing 20
mM potassium phosphate in the absence and in the
presence of 20 wt% EG, 20 wt% PEG 8000 and 10 wt% PEG
8000. At all studied conditions, the melting profiles show
clear transition at 295 nm (Figure 6A). CD spectra at
different temperatures in the same buffer show isosbestic points
near 225 and 250 nm, and linear correlations between the
signal changes at 265 and 243 nm during the melting
(Figure 6). These results clearly indicate a two-state transition
of the (un)folding equilibria of M2 G-quadruplex.
Thermodynamic parameters, i.e. enthalpy ( H◦) and entropy
( S◦) changes and stability at 25◦C ( G◦25) for the
formation of the G-quadruplex calculated by fitting the melting
transitions are shown in Table 1. Addition of either EG or
PEG 8000 stabilizes the M2 G-quadruplex due to favorable
enthalpy contribution. Previous reports have demonstrated
the dominant effect of dehydration for stabilization of not
only G-quadruplexes, but also other nucleic acid structures
). The stabilization of the M2 G-quadruplex by EG
is likely caused by an osmolyte effect of EG involving
dehydration upon formation of the G-quadruplex, because the
water activity is clearly reduced by addition of EG.
Interestingly, PEG 8000 stabilizes the M2 G-quadruplex to a greater
extent than EG at 20 wt%, although PEG 8000 does not
behave as an osmolyte and does not reduce the water
activity comparing to EG (Supplementary Table S4). The
energy difference in the presence and in the absence of cosolute
( G◦25) is more than twofold higher in the presence of 20
wt% PEG 8000 ( G◦25 = 7.37 kcal mol−1) comparing to
the same weight percent of EG ( G◦25 = 3.43 kcal mol−1).
The stability of the M2 G-quadruplex in the presence of 20
wt% EG is comparable with that in the presence of 10 wt%
PEG 8000. As dehydration is not the prime origin of PEG
8000-induced stabilization, the observed stabilization
corresponds to other factors in solution or direct interaction
between PEG 8000 and M2 G-quadruplex.
UV melting analyses were performed in a buffer
containing 50 mM MES–LiOH (pH 7) in the absence and in the
presence of 20 wt% cosolute with various concentrations
of KCl to evaluate an effect of cation concentration on the
G-quadruplex stability (Supplementary Figure S4a-c).
Supplementary Table S5 shows thermodynamic parameters
calculated from the UV meting curves. At a range of potassium
ion concentrations, at which M2 G-quadruplex showed
clear melting transition between 20 and 95◦C, G◦25 of
the M2 G-quadruplex and natural logarithm value of the
potassium ion concentration (ln[KCl]) showed clear linear
correlation (Supplementary Figure S4d). Slope value of the
plots reflects a number of potassium ions involved in the
G-quadruplex formation or folding cooperativity of the
Gquadruplex in response to changing concentration of
potassium ions (
). The absolute slope values increased in the
presence of cosolutes, indicating that upon folding of M2
Gquadruplex under the molecular crowding conditions the
uptake of potassium ions is higher than under the diluted
condition. Thus, it is expected that in the presence of 100
mM potassium ions, at which we analysed NMR structure,
the difference of G-quadruplex stability between the diluted
and crowding conditions are larger than those reported in
Table 1. UV melting profiles also indicate that the
potassium ion concentration, at which a half of oligonucleotide
forms G-quadruplex at 25◦C ( G◦25 = 0), is lower in the
presence of cosolutes comparing to the diluted condition
(Supplementary Figure S4D). These results suggest that not
only cooperativity but also the observed binding affinity of
potassium ions increases under the crowding condition.
Importantly, interactions between potassium ions and M2
Gquadruplex seems more efficiently enhanced by PEG 8000,
because the absolute slope value in G◦25 vs. ln[KCl] plots is
larger, while lower potassium ion concentration is required
for the G-quadruplex folding in comparison to EG.
Overhanging residues of the M2 G-quadruplex in the role of antenna for cosolute-induced changes
Upon addition of 20 wt% EG changes in 1H NMR chemical
shifts of methyl and aromatic protons of T1-G19 residues of
the M2 G-quadruplex are observed in the range between
0.01 and 0.03 ppm (Figure 7 and Supplementary Table S6).
Noteworthy, these values are very close to the limits of the
experimental error. On the other hand, there is a significant
downfield shift of methyl as well as H6 protons of T20 by
0.08 and 0.11 ppm, respectively, indicating that their local
environments are changed considerably upon addition of 20
wt% EG. These changes may result from decrease in water
activity in the presence of 20 wt% EG and/or associated
conformational change of residue T20. It is noteworthy that
coupled alteration of M2 G-quadruplex structure and
water network that are induced by the high concentrations of
EG seem likely, especially as careful inspection of NOESY
spectra recorded at mixing times in the range between 80
and 300 ms did not reveal any long-lived interactions
between the M2 G-quadruplex and EG.
Perturbations in 1H NMR chemical shifts upon addition
of 10 or 20 wt% PEG 8000 are evident for all residues
corresponding to the M2 G-quadruplex (Figure 7 and
Supplementary Table S6). Notably, the extent of 1H NMR
chemical shift changes correlates with the increased concentration
of PEG 8000 (Supplementary Figure S5). The effect is by far
most pronounced for residue T20. Moreover, consequent
with addition of 10 wt% and 20 wt% PEG 8000 the changes
in 1H NMR chemical shift of T20 H6 aromatic proton are
0.43 and 0.52 ppm, respectively. The substantial PEG
8000induced changes in the chemical environment at the 3 -end
of the M2 G-quadruplex are further demonstrated by the
large and gradual downfield shift of T20 methyl 1H NMR
signal. Interestingly, at 20 wt% PEG 8000, the aromatic
Values are average ± s.d. from at least triplicated experiments.
tons of G5, G10, G14 and G19 residues comprising the
G-quartet positioned in vicinity of T20 are clearly shifted
downfield by 0.13, 0.07, 0.07 and 0.13 ppm, respectively. In
comparison, perturbations in 1H NMR chemical shifts for
aromatic protons of guanine residues in the G3–G8–G12–
G17 and G4–G9–G13–G18 quartets are in the range
between –0.02 and 0.04 ppm and thus considerably smaller
with respect to the G5–G10–G14–G19 quartet.
Noteworthy, addition of 20 wt% EG (in contrast to PEG 8000) does
not lead to notable deshielding of 1H NMR signals of
guanine residues, which may reflect differences between
EGand PEG 8000-M2 G-quadruplex interactions. The
addition of 10 wt% PEG 8000 gives rise to minor perturbations
in methyl and aromatic 1H NMR chemical shifts (
below 0.06 ppm) of the 5 -end overhanging residues of the M2
G-quadruplex. Interestingly, however, the increase of PEG
8000 to 20 wt% results in considerable downfield shifts of
T1 methyl and H6 as well as of A2 H8 1H NMR signal,
which are close or above 0.10 ppm with respect to
cosolutefree condition. This indicates that PEG 8000, at least at 20
wt%, induces substantial changes in the chemical
environment not only at the 3 , but also at the 5 -end
overhanging region of the M2 G-quadruplex. Altogether, the
analysis of 1H NMR chemical shifts perturbation for the M2
G-quadruplex upon addition of PEG 8000 indicates
specificity of cosolute-induced effects, which are minor at the
core of the structure, significant at the 5 -end and profound
at the 3 -end of the structure. In this regard, it is noteworthy
that perusal of NOESY spectra does not reveal any
crosspeaks in support to direct binding of PEG 8000 to the M2
EG and PEG 8000 modulate M2 G-quadruplex structure differently
We calculated the solution-state structure of the M2
Gquadruplex formed in the presence of potassium ions and
absence of cosolutes with the use of 427 NOE-derived
distance restraints together with 97 torsion angle and 24
hydrogen-bond restraints (Figure 8A). Well-converged ten
representative structures exhibit a pairwise heavy atom
RMSD of 0.89 A˚ (structural statistics are given in
Table 2). All the guanine residues involved in G3–G8–G12–
G17, G4–G9–G13–G18 and G5–G10–G14–G19 quartets
exhibit anti glycosidic conformations. The three G-quartets
are stacked on top of each other and represent the core of
the structure. G-quartets are not perfectly planar as
several guanines, in particular G4, G8, G9, G10, G14 and
G17 exhibit orientation with O6 being slightly bent toward
the 3 -end (Supplementary Figure S6). The 5 -end
overhanging residues T1 and A2 are positioned on top of the
central cation cavity (Figure 8D and E). Among the
ensemble of the representative structures certain
conformational freedom is observed for T1, whereas conformation
of A2 is very well converged. The purine ring of A2 is
parallel with respect to the plane of the nearby
G3-G8-G12G17 quartet. The 3 -end is well defined and exhibits T20
stacked over G19. A6 and C7 form propeller-type loop
thereby linking G5 and G8 residues of the 5 - and 3 -end
Gquartets, respectively. Orientation of A6 is well defined, with
its purine ring inclined by ca. 45◦ with respect to the plane
of the nearby central G4–G9–G13–G18 quartet
(Supplementary Figure S7). C7 exhibits more conformational
freedom in comparison to A6. The central loop comprises C11
and bridges the G8–G9–G10 and G12–G13–G14 stems
of the M2 G-quadruplex via propeller-type arrangement.
The loose conformation of C11, together with its
orientation toward solvent is reminiscent of previous reports on
single-nucleotide propeller-type loops in parallel stranded
G-quadruplex structures (Supplementary Figure S8) (
). Structures of C15 and A16 are well defined and form
the third propeller-type loop. Moreover, C15 is oriented
away from the core of the structure toward the solvent,
whereas A16 is inserted into the groove formed by G12–G14
and G17–G19 stems (Supplementary Figure S9). The M2
G-quadruplex is characterized by the average groove width
of 11.1 (± 0.5) A˚ which is thus slightly broader with respect
to the parallel-stranded N-myc (20) G-quadruplex (10.7 ±
0.5 A˚ ) exhibiting three single-residue propeller-type loops
and three G-quartets in the core of the structure.
High-resolution structure of M2 G-quadruplex in the
presence of 20 wt% EG was calculated using 447
NOEderived distance restraints. Ten representative structures of
parallel stranded M2 G-quadruplex with the three stacked
G-quartets in the core exhibit a pairwise heavy atom RMSD
of 1.27 A˚ (Figure 8B; structural statistics are given in
Table 3). This structure exhibits an average groove width of
11.0 (± 0.6) A˚. All guanine residues adopt anti glycosidic
conformation. T1 and more so A2 at the 5 -end overhang
region exhibit well-defined conformations and together cap
the nearby G3–G8–G12–G17 quartet (Figure 8D and E).
T20 at the 3 -end exhibits a well-defined conformation and
is stacked on the pyrimidine moiety of G19 (Figure 8F and
G). In the first propeller-type loop of the M2 G-quadruplex
in the presence of 20 wt% EG A6 exhibits a well-defined
conformation, while conformation of C7 is poorly defined.
C11 in the second propeller-type loop occupies the groove
between G8–G10 and G12–G14 stems with the base moiety
oriented inward the G5–G10–G14–G19 quartet
(Supplementary Figure S8). Notably, eight among ensemble of ten
representative structures exhibit C11 with a well-converged
conformation. The two remaining structures are diverse,
altogether indicating that conformation of C11 is not
defined precisely. In contrary, the conformation of the third
propeller-type loop is very well-defined with both residues,
C15 and A16, being well converged among the ensemble of
ten representative structures.
There is great resemblance between the calculated M2
Gquadruplex structures in the absence and in the presence
of 20 wt% EG. Structures share orientations of residues
at the 5 -end (Figure 8), albeit the ones corresponding to
cosolute-free condition exhibit A2 seemingly slightly closer
to the nearby G3–G8–G12–G17 quartet. Further, cores of
the structures in the absence and in the presence of 20
wt% EG grossly match, with some deviations in
conformations of guanine residues. In particular, in the presence of
20 wt% EG there is an out-of-plane bending of G4, G10,
G14, G17, G18 and G19 toward the 5 -end and shifting of
G4, G9, G12, G17 and G18 within the G-quartet planes
in comparison to the structures in the absence of
cosolutes (Supplementary Figure S6). These variances, however,
could in principle be ascribed in the range of ‘breathing’
motions within G-quartets that are commonly observed in
G-quadruplexes. The first and the third two-residue loops
A6-C7 and C15-A16, respectively, appear very similar in
the absence and in the presence of 20 wt% EG, with the
exception that the cosolute imposes a slightly smaller
inclination of A6 with respect to G4–G9–G13–G18 quartet plane,
i.e. ca. 30◦ versus 45◦ (Supplementary Figure S7). On the
other hand, a substantial structural change can be assigned
to the presence of EG for the central one-residue
propellertype loop. Moreover, in eight out of ten representative
structures of the M2 G-quadruplex in the presence of 20 wt%
EG residue C11 is oriented toward the G5–G10–G14–G19
quartet and not aside the structural core as in the
cosolutefree condition (Supplementary Figure S8). Coupled with
the variance in C11 orientation the sugar-phosphate
backbone in the G8–G13 segment is different between the
structures in the absence and in the presence of 20 wt% EG.
Notably, however, orientation of C11 is not firmly restrained
by the experimental data in either of the conditions, thus
suggesting that EG exhibits only a limited impact on
orientation of loop residues. Yet, there exists a clear and
consistent distinction between the M2 G-quadruplex structures
in the presence and absence of EG and it is located at the
3 -ends of the structures (Figure 8F–G). In the absence of
cosolutes T20 stacks over pyrimidine and imidazole
moieties of G19, whereas in the presence of EG the interaction
interface comprises almost exclusively pyrimidine moiety of
G19. Additionally, the presence of EG is coupled with
position of T20 closer to the top of the central cation channel
of M2 G-quadruplex. Notably however, the overall distance
between T20 and G19 appears similar in the absence and
in the presence of EG, due to EG-induced change in their
backbone conformations as well as out of plane bending of
G19 toward the 5 -end. It is worth noting that for individual
conditions, i.e. in the absence and in the presence of 20 wt%
EG, the conformation of T20 is very well converged among
the ensembles of the calculated structures. The prominent
conformational changes of T20 in response to the presence
of EG are consistent with the major perturbations of its 1H
NMR chemical shifts (Figure 7). On the other hand,
EGinduced changes in 1H NMR chemical shifts for residues
in T1-A2 and G10–C11–G12 segments are only minor,
although slight deviations in their conformations were
observed with respect to the calculated structure in the absence
of cosolutes. Overall, the NMR-based structural
comparison suggests that the differences in biophysical properties of
the M2 G-quadruplex in the absence and in the presence of
EG (vide supra) are coupled to T20 conformational changes,
while effect is considerably smaller for the three loops and
the 5 -end overhang.
The high-resolution structure of parallel-stranded M2
G-quadruplex in the presence of 10 wt% PEG 8000 was
calculated with the use of 459 NOE-derived distance
restraints, 97 torsion angle restraints and 24 hydrogen-bond
restraints. The ten representative structures exhibiting a
pairwise heavy atom RMSD of 1.06 A˚ are shown in
Figure 8C (structural statistics are given in Table 4). Twelve
guanine residues, all characterized by anti glycosidic torsion
angle values, form three stacked G-quartets at the M2
Gquadruplex core. The average groove width is 11.0 (±0.7) A˚ ,
matching well to the structures in cosolute-free and 20 wt%
EG conditions. The G3–G8–G12–G17 quartet is capped
with the 5 -overhang residues T1 and A2, which are
successively stacked, whereby A2 is positioned partly above G3
and partly above the central cation channel. T1 and A2 are
both well-converged among the ensemble of the ten
representative structures. The conformation of T20 at the 3
end is well-defined. Interestingly, T20 is oriented away from
the G-quartet ‘platform’ and is positioned in extension of
the groove defined by G3–G5 and G17–G19 stems, with its
sugar moiety facing the solvent. A6, which is the first of the
two residues in the first propeller-type loop, is well-defined
and exhibits purine moiety inserted in the groove in the G3–
G5 and G8–G10 region. Additionally, the purine base of A6
is slightly (ca. 30◦) inclined with respect to the central G4–
G9-G13–G18 quartet (Supplementary Figure S7).
Conformation of C7 is rather flexible. The second propeller-type
loop is well-defined and exhibits C11 residue oriented
toward the solution. C15 in the third propeller-type loop is
positioned away from the core of the structure enabling
insertion of A16 into the groove defined by G12–G14 and
G17–G19 stems. Among the ten representative structures of
the M2 G-quadruplex in the presence of 10 wt% PEG 8000,
orientation of A16 congregates into two clusters
(Supplementary Figure S9D and E). In the cluster of seven
structures the A16 purine ring caps the groove defined by G12–
G14 and G17–G19 stems. The cluster of the remaining three
structures is defined by A16 being closer to the central G4–
G9–G13–G18 quartet and with plane of its base moiety
appearing to intersect the nearby groove of G12–G14 and
In the presence of PEG 8000 the conformation of T20
at the 3 -end is very different with respect to the structure
in the absence of cosolutes (Figure 8F and G). In
cosolutefree conditions the sugar-phosphate backbone progression
in the G17-T20 segments of the M2 G-quadruplex is
uniform, enabling T20 to stack on G19. In comparison, the
addition of PEG 8000 results in T20 sugar moiety to
orient closer to the groove defined by G12–G14 and G17–G19
stems, while its pyrimidine moiety is pushed aside of G5–
G10–G14–G19 quartet. The conformational change of T20
corresponds to the largest difference between the structures
in the absence and in the presence of 10 wt% PEG 8000 and
thus corroborates the results of 1H NMR chemical shift
perturbation analysis (Figure 7). In addition, the presence of
10 wt% PEG 8000 very modestly affects the conformation
of the 5 -end of the M2 G-quadruplex in that T1 is slightly
closer to A2. The conformation of T1 is more converged
among the structures in the presence of 10 wt% PEG 8000,
altogether suggesting more constrained disposition of the
T1-A2 ‘cap’ over the nearby G-quartet in comparison to
the structure in cosolute-free condition. Although, guanine
residues involved in the core of the M2 G-quadruplex in the
presence of 10 wt% PEG 8000 are not superimposable to
the structures in the absence of cosolutes perfectly
(Supplementary Figure S6), the differences are minor. In this
regard, the distinction is even less prominent than between the
structures in cosolute-free and EG conditions. PEG
8000induced structural changes in the first propeller-type loop
are confined to A6, whose purine moiety appears slightly
less inclined with respect to the plane of G4–G9–G13–G18
quartet. Interestingly, the conformation of A6 is highly
similar in the presence of 10 wt% PEG 8000 and 20 wt% EG
(Supplementary Figure S7). The disposition of the second
loop comprising C11 is not significantly affected by the
presence of 10 wt% PEG 8000. However, PEG 8000 appears
to promote moderate flexibility of the third propeller-type
loop comprising C15 and A16. Furthermore, only three
among the ensemble of the ten representative structures in
the presence of 10 wt% PEG 8000 exhibit C15 and A16 in
a disposition matching well to the conformation observed
in the structures in the absence of cosolutes and in the
presence of 20 wt% EG. However, the significance of the PEG
8000-induced variable disposition of C15 and A16 in the
calculated structures is questionable, especially considering
that only marginal 1H NMR chemical shifts changes are
observed for the C15-A16 segment upon addition of PEG
8000. Overall, it seems that 10 wt% PEG 8000 only modestly
effects disposition of loops and the 5 -end residues, while it
has a significant impact on the conformation of T20 at the
3 -end of the M2 G-quadruplex.
In order to correlate the structural differences with
intrinsic energy of the M2 G-quadruplexes under the three
different conditions we performed electronic state calculations
(Supplementary Data). Fragment molecular orbital (FMO)
method in vacuum condition was utilized to carry out the
interfragment interaction energy (IFIE) analysis to detect
the direct molecular interactions between fragments
(Supplementary Figure S10 and Supplementary Tables S7 to
). The orientation of C11 toward the G5–G10–
G14–G19 quartet in the presence of 20 wt% EG contributes
to lower IFIE of the M2 G-quadruplex, while the
orientation toward the bulk solution observed in the presence of
10 wt% PEG 8000 and cosolute-free condition increases the
IFIE. The position of A16 as a cap over the groove between
G12–G14 and G17–G19 stems does not contribute to the
IFIE as much as when it is located closer to the central G4–
G9–G13–G18 quartet. Reduction of IFIE involving residue
T20 follows the following order: 20 wt% EG > cosolute-free
> 10 wt% PEG 8000. According to the above contributions,
the overall IFIE of the M2 G-quadruplex is the highest in
the presence of 20 wt% EG and is followed by cosolute-free
and 10 wt% PEG 8000 conditions (Supplementary Table
S10). However, due to considerably large standard
deviations, the energy differences cannot be clearly distinguished
between the structures under the three conditions.
Molecular crowding conditions with high concentration of
cosolutes potentially alter the folding and thermodynamic
stability of nucleic acids through interplay of various
factors, such as reduced water activity, dielectric constant and
also excluded volume effect that depend on chemical
properties and molecular weight of the cosolutes (
). In the
case of G-quadruplexes, stabilization and monomorphic
property of their structures in the presence of the
crowding cosolutes are caused likely by dehydration during their
formation under the conditions of reduced water activity
). Heddi et al. reported the first and one of the rare
insights into high-resolution G-quadruplex structures in
the presence of PEG (25). Moreover, these authors
concluded that the conformational transition from
polymorphic G-quadruplex mixture present under diluted solution
to monomorphic parallel topology in the presence of PEG
is predominantly caused by water depletion. However, apart
from the indirect effects of the crowding cosolutes, also
direct binding of PEG to DNA G-quadruplex should be
noted. For example, not only the interactions between
hydrophilic PEG moieties and DNA sugar-phosphate
backbone, but also PEG amphiphilic nature and in this regard
its organic-like properties were pointed out in deducing the
driving force for PEG-DNA interactions (
). In fact, it is
the hydrophobic counterparts that were perceived to note
‘non-inertness’ of PEG with respect to biomolecules (
With regards to investigating structural properties of G-rich
DNA, there are compelling arguments against the use of
PEG as a molecular crowding agent due to its direct
impact on G-quadruplex stability and topology (
). It is
likely that there are different views on appropriateness of
PEG as an inert crowding cosolute depending on its
molecular size. Indeed, the solution properties very much depend
on the length of PEG as evident from the water activities
in buffers containing 20 wt% EG and PEG 8000
(Supplementary Table S4). Nevertheless, various polymeric
ethylene glycol derivatives are still used in order to impose
celllike environment and thereupon perform physicochemical
analyses of DNA G-quadruplexes. Notably, however,
calculation and comparison of high-resolution structures in
the presence of different cosolutes, which may behave as
osmolyte and/or putative crowding agent were not reported.
In this regard, generalization of PEG-DNA G-quadruplex
interactions and the biological relevance of G-rich DNA
features in solutions comprising PEGs remain unrewarding
because very scarce structural data in the presence of these
cosolutes limits in-depth comprehension. Thus, structural
details underlying EG- and PEG 8000-M2 G-quadruplex
interactions will be useful to constructively discuss the
issue and further our understanding. This is especially
relevant in light of the recent reports on nucleic acids-ligand
interactions in different environments, which unrevealed
the discrepancy of PEG-crowding systems and cell extracts,
thereby further marking the importance of subtle
heteromolecular, i.e. DNA/RNA-protein interactions, which are
generally absent in cell-mimicking systems (
At the herein used experimental setups M2 G-quadruplex
exhibits preserved parallel topology both in the absence and
in the presence of cosolutes as demonstrated by
complementary spectroscopic methods. Since M2 may form
highorder structures in the presence of 20 wt% PEG 8000, which
mists and complicates NMR characterization, we analysed
detailed structure of the M2 G-quadruplex in the absence
of cosolute and in the presence of 20 wt% EG and 10 wt%
PEG 8000. Importantly, the overall structure of the M2
Gquadruplex, especially the core G-quartets region, did not
show considerable changes in the absence and in the
presence of the cosolutes. In the case of loop nucleotides,
certain differences were observed such as inward orientation
of C11 in the presence of 20 wt% EG and capping-like
orientation of A16 with respect to the groove defined by G12–
G14 and G17–G19 segments in the presence of 10 wt% PEG
8000 (Supplementary Figures S8 and S9). In contrast,
considerable and distinct structural changes were observed for
residue T20 that are corroborated by perturbations in 1H
NMR chemical shifts in the presence of both 20 wt% EG
and 10 wt% PEG 8000 with respect to cosolute-free
condition (Figure 8).
In the presence of 20 wt% EG, perturbations in the 1H
NMR chemical shifts at the 3 -end region of the M2
Gquadruplex can be partly attributed to the fact that outer
G-quartets are typically much more hydrophobic with
respect to other G-quadruplex regions. In general, due to
the hydrophobicity, the 5 - and 3 -flanking nucleobases in
the G-quadruplexes tend to stack on the outer G-quartets.
This kind of stacking interaction of flanking nucleobases
that contributes to overall stability is also reported in
single nucleotide overhanging regions of DNA and RNA
). In the duplexes, the dangling end is hydrated
more than the blunt end and destabilized by osmotic stress
caused by a cosolute, which lowers water activity (57). In the
herein studied system the addition of 20 wt% EG is
accompanied by dehydration of residue T20 furthermore leading
to its orientation toward the cation central-channel cavity.
Thereby, the electrostatic repulsion between T20 and G5–
G10–G14–G19 quartet is neutralized in favor of the
stacking interactions. On the other hand, T1 and A2 at the 5 -end
keep their conformation due to the stacking interactions
impregnable to the osmotic stress. Moreover, the 5 -end
adenine (purine) residue interacts with the outer G-quartet
more tightly compared to the 3 -end thymine (pyrimidine)
residue through stronger stacking interactions. The
conformational change of C11 toward the groove in the region
between G10 and G14 and closer to G5-G10-G14-G19
quartet is coupled with the dehydration effect of EG.
Noteworthy, in the presence of PEG 8000 residue C11 retains its
disposition toward the bulk solution.
The UV-melting experiments clearly demonstrate that
the EG and PEG 8000 significantly stabilize the M2
Gquadruplex (Supplementary Figure S4 and Supplementary
Table S5). These results together with IFIE analysis suggest
that the intrinsic structural interaction energy itself does not
correlate to the actual stability of the M2 G-quadruplex in
the presence of cosolutes. Thus, the stabilization of the M2
G-quadruplex by EG and PEG 8000 is related to
extrinsic factors. As mentioned above, EG behaves as osmolyte,
which reduces water activity, rather than macromolecular
crowding cosolute (Supplementary Table S4). The reduced
water activity in the presence of 20 wt% EG is expected to be
coupled with conformational changes of C11 and T20, and
overall stabilization of the M2 G-quadruplex through
intrinsic dehydration during formation of the core G-quartets.
In addition, reduced dielectric constant caused by the EG
might also contribute to the stabilization, although such
effect is expected to be more prominent in the case of PEG
In the presence of PEG 8000, not only the 1H NMR
chemical signals for residue T20, but also aromatic protons
of guanines in G5–G10–G14–G19 quartet are significantly
de-shielded, which is not the case in the presence of EG.
These results suggest resolved stacking interactions between
residue T20 and the outer G-quartet in the presence of PEG
8000, and different local environment of the 3 -end residue
with respect to the presence of 20 wt% EG (and in the
absence of cosolutes). Additionally, as depicted by the
calculated structures in the presence of PEG 8000, T20 is oriented
toward the groove region and is unstacked from G5–G10–
G14–G19 guartet suggesting that the G-quartet platform
is thereby available for interactions with the cosolute.
Recently, Buscaglia et al. implied preferential binding of the
high molecular-weight PEGs to external G-quartets.
Additionally, we have also shown that oligoethylene glycols
stabilize G-quadruplexes through CH- and lone pair-
). Thus, the structural change of T20 in the
presence of PEG 8000 is potentially affected by direct
interaction between PEG 8000 and M2 G-quadruplex. In this
regard, scrutiny of cosolute-M2 G-quadruplex interactions
and in particular the lack of NOE cross-peaks
corresponding to close positioning of PEG 8000 to M2 G-quadruplex,
i.e. within 5 A˚, indicates the absence of long-lived
intermolecular interactions. However, these observations do not
disprove subsistence of direct DNA binding by PEG 8000.
Previous reports estimated the dissociation constant KB of
PEG to G-quadruplex forming DNA at molar to sub-molar
). By considering the molar concentrations of
10 wt% PEG 8000 and DNA at approximately 12 and 0.2
mM, respectively, the lack of NOE cross-peaks for their
intermolecular interactions might be interpreted by fast
exchange on NMR timescale of the PEG 8000 between bulk
solution and DNA-binding platform(s) due to the weak
Notably, PEG 8000 is not an osmolyte and the water
activity in the presence of 20 wt% PEG 8000 is considerably
higher than in the presence of 20 wt% EG and is close to
cosolute-free conditions (Supplementary Table S4). Thus, it
is considered that the water activity cannot solely account
for the stabilization of the M2 G-quadruplex. One of the
potential factors for stabilization of the M2 G-quadruplex
is excluded volume effect provided by PEG 8000. Excluded
volume is a volume occupied by a molecule in a system
and therefore inaccessible for other molecules. For
considering the degree of the excluded volume effect it is
important to evaluate hydrodynamic radius of the molecules in
the system that defines their hydrodynamic volume in
). Large excluded volume is provided to a solute
molecule from co-existing crowding agent when they have
similar hydrodynamic volumes (
). Large sized PEGs
have previously been demonstrated to impose excluded
volume effect against similar sized nucleic acids, which
facilitates compaction of their structures including
stabilization of canonical duplex and tertiary structures (
this regard, stabilization of the M2 G-quadruplex by PEG
8000 is expected. In addition to imposing the excluded
volume, large sized PEGs such as PEG 8000 reduce
dielectric constant of the solution more than EG
(Supplementary Table S4), which could be also a reason for facilitation
of ribozyme activity in the presence of PEG (64).
Reduction of the dielectric constant of the solution should
impact the G-quadruplex stability mediated by interactions
between an ion and G-quadruplex. In fact, it has been
shown that decrease in dielectric constant followed by
addition of small organic solvents leads to increased
stability of G-quadruplexes via favored electrostatic interactions
). The plots of G◦25 versus ln[KCl] (Supplementary
Figure S4) showed larger cooperativity and lower
concentration of potassium ions required for the folding of M2
Gquadruplex in the presence of 20 wt% PEG 8000 compared
to 20 wt% EG. EG also reduces dielectric constant and thus
the cooperativity of potassium ion for the G-quadruplex
folding in the presence of EG was higher than in the absence
In conclusion, structural details on the M2 G-quadruplex
in the presence and absence of crowding cosolutes reveal
that the variances mainly concern T20 residue at the 3 -end.
Furthermore, we show that PEG 8000 interacts with M2
G-quadruplex at its 3 -end, which is consistent with the
suggested direct interaction between large size PEG and outer
G-quartets. However, our complementary approach
indicates that the interactions between PEG 8000 and the M2
G-quadruplex are weak and in this regard the stabilization
contribution might be related also to the changes in
solvation continuum and excluded volume by PEG 8000. On the
other hand, the stability of M2 G-quadruplex is increased
by EG mainly due to dehydration effect.
Data has been deposited in the Protein Data Bank (https:
//www.rcsb.org/) and Biological Magnetic Resonance Data
Bank (http://www.bmrb.wisc.edu/) under the following
accession numbers: PDB 5NYS, BMRB 34135, PDB 5NYT,
BMRB 34136, PDB 5NYU, BMRB 34137.
Supplementary Data are available at NAR Online.
Slovenian Research Agency (ARRS) [P1-0242, J1-6733];
CERIC-ERIC Consortium for the access to experimental
facilities and financial support; Japan Society for the
Promotion of Science (JSPS) and Slovenian Research Agency
[BI-JP/15-17-002] under JSPS Bilateral Joint Research
Program; Grant- in-Aid for Scientific Research on
Innovative Areas ‘Chemistry for Multimolecular Crowding
Biosystems’ (JSPS KAKENHI) [JP17H06351], for Scientific
Research, Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan; MEXT-Supported Program
for the Strategic Research Foundation at Private
Universities (2014–2019), Japan; Hirao Taro; Okazaki Kazuo
Foundation of KONAN GAKUEN for Advanced Scientific
Research; Chubei Itoh Foundation. Funding for open access
charge: Slovenian Research Agency (ARRS) [P1-0242,
Conflict of interest statement. None declared.
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