2-O-Methyl-8-methylguanosine as a Z-Form RNA stabilizer for structural and functional study of Z-RNA

Article abstract of DOI:10.3390/molecules23102572

In contrast to Z-DNA that was stabilized and well-studied for its structure by chemical approaches, the stabilization and structural study of Z-RNA remains a challenge. In this study, we developed a Z-form RNA stabilizer m⁸Gm, and demonstrated that incorporation of m⁸Gm into RNA can markedly stabilize the Z-RNA at low salt conditions. Using the m⁸Gm-contained Z-RNA, we determined the structure of Z-RNA and investigated the interaction of protein and Z-RNA.

Full text of DOI:10.3390/molecules23102572

molecules
Article
0
2
-O-Methyl-8-methylguanosine as a Z-Form RNA
Stabilizer for Structural and Functional Study
of Z-RNA
1
1
, Chao-Da Xiao 1,2, Hong-Liang Bao 1 and
Thananjeyan Balasubramaniyam , Takumi Ishizuka
Yan Xu *
1
,
1
Division of Chemistry, Department of Medical Sciences, University of Miyazaki, 5200 Kihara, Kiyotake,
Miyazaki 889-1692, Japan; balasubramaniyam_thananjeyan@med.miyazaki-u.ac.jp (T.B.);
tishizuka@med.miyazaki-u.ac.jp (T.I.); choutatsu_shou@med.miyazaki-u.ac.jp (C.-D.X.);
xiaobujiao@163.com (H.-L.B.)
The key Laboratory of Optimal Utilization of Natural Medicine Resources, School of Pharmaceutical
Sciences, Guizhou Medical University, University Town, Guian New District, Guiyang 550025, China
Correspondence: xuyan@med.miyazaki-u.ac.jp; Tel.: +81-985-85-0993
2
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 5 September 2018; Accepted: 7 October 2018; Published: 9 October 2018
Abstract: In contrast to Z-DNA that was stabilized and well-studied for its structure by chemical
approaches, the stabilization and structural study of Z-RNA remains a challenge. In this study, we
8
8
developed a Z-form RNA stabilizer m Gm, and demonstrated that incorporation of m Gm into
8
RNA can markedly stabilize the Z-RNA at low salt conditions. Using the m Gm-contained Z-RNA,
we determined the structure of Z-RNA and investigated the interaction of protein and Z-RNA.
Keywords: Z-RNA structure; synthesis of oligonucleotide; NMR; circular dichroism
1
. Introduction
The RNA helixes mostly adopt a right-handed A-from geometry. Another RNA conformation is
characteristic of left-handed RNA having alternating CG base pairs [
of RNA-editing enzyme ADAR1, has been found to specifically bind to Z-DNA and Z-RNA [
It has been reported that Z-RNA participates in the interferon-response pathway [10 11] or viral
inhibition [10 12]. The physical, chemical and spectral data on Z-RNA have been collected, whereas
1,
2
]. Z
α
, a Z-form-binding domain
3–9].
,
–
the biological role of Z-RNA has long been in question due to the difficulty of obtaining stable Z-RNAs
under physiological salt conditions. For example, an A- to Z-RNA transition requires a very high salt
concentration, e.g., 6 M NaClO [1,2].
4
Several studies have been reported on incorporating modified nucleic acid residues for the
stabilization of Z-DNA [13–21]. Notably modified guanine residues highly favor the formation of
Z-DNA. We have previously reported that the methylation of the C8 position of guanine significantly
stabilized the Z-DNA because the modification by methylation was favorable for the syn conformation
of the nucleobases [17]. Based on the results of these past studies, we have now developed a Z-form
RNA stabilizer that stabilizes Z-RNA under physiological salt conditions. We designed and synthesized
0
8
a 2 -O-methyl-8-methyl guanosine (m Gm) by insertion of a methyl group at the C8 position of
2
0
8
-O-methyl guanosine. We found that incorporation of the m Gm in the RNA dramatically stabilized
the Z-RNA, even under physiological salt concentrations, and facilitated the A- to Z-RNA transition,
even for AU-containing sequences that do not favor the formation of Z-RNA. We then determined the
8
solution structure of r(CGC[m Gm]CG) . This allowed us to see the effect of the incorporation of a
2
8
m Gm on RNA and allowed us to understand the Z-RNA structure. The Z-RNA stabilizer can also be
used to investigate the interaction of the Zα domain and Z-RNA.
Molecules 2018, 23, 2572; doi:10.3390/molecules23102572
www.mdpi.com/journal/molecules

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2
. Results and Discussion
8
We synthesized the m Gm-incorporating oligonucleotides by phosphoramidite chemistry, using
0
2
-O-methyl guanosine (Schemes S1, S2 and Figures S1–S7). The induction of a methyl group in the C8
8
position highly favors the syn conformation. These structural features can lead the m Gm to greatly
0
stabilize Z-RNA. Using nuclear Overhauser effect (NOE) between C1 H and 8CH we confirmed the
3,
8
syn conformation of m Gm (Figure S8).
Circular dichroism (CD) spectroscopy has been used to study the Z-form conformation [17
A-RNA, a negative Cotton effect appears around 295 nm, whereas in Z-RNA, a more positive band
appears at 280 nm [23 24]. Thus, we performed CD spectroscopy experiments to study the conformation
,22].
,
using various NaClO concentrations.
4
We used the CD spectroscopy to examine the A-Z transition under various salt concentrations.
Native RNA r(CGCGCG) in A-form does not transfer to the Z-form, even in the presence of
2
8
8
3
M NaClO (Figure 1a). m Gm-incorporated r(CGC[m Gm]CG) showed a Z-form CD spectrum
4 2
with increasing concentrations of NaClO . (Figure 1b). The midpoint NaClO concentration
4
4
8
for r(CGC[m Gm]CG) was 880 mM, lower than that of the native RNA (4090 mM) (Table 1).
2
8
8
8
r(C[m Gm]C[m Gm]CG) containing two m Gms greatly stabilized the Z-form, showing a Z-form CD
2
spectrum even in the presence of 50 mM NaClO (Figure S9) at a lower physiological salt concentration,
4
and the midpoint was 100 mM (Table 1).
8
The incorporation of m Gm can stabilize the Z-form containing an AU base pair. It showed
the typical Z-form with 2360 mM midpoint compared to the native RNA (Figure S9 and Table 1).
8
Octamer RNA containing four m Gms in double strands also effectively stabilized the Z-form with
8
50 mM midpoint.
8
8
We designed a short duplex containing only one modified base, r(C[m Gm]CGU[m Gm]CG)/
8
r(CGCACG), in which the underlined sequences can form a duplex containing only one m Gm base.
We compared its stability with native RNA r(CGCGUGCG)/r(CGCACG). Although the 6 base pairs
duplex containing an AU base pair does not favor the formation of Z-RNA, it facilitated the A- to
Z-RNA transition as compared to native 6 base pairs RNA (Figure S10). The midpoint for RNA
containing one m Gm was 4000 mM, lower than that of the native RNA (>6000 mM) (Figure S10),
indicating that only one m Gm can stabilize the Z-form, indicating that only one m Gm can stabilize
the Z-form.
8
8
8
8
8
Figure 1. CD spectra of native RNA r(CGCGCG) (
(
a
) and m Gm-contained r(CGC[m Gm]CG) (
b
)
2
2
◦
0.15 mM base concentration) at 10 C with various NaClO concentrations in 5 mM sodium phosphate
4
buffer (pH 7.0).

Molecules 2018, 23, 2572
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Table 1. Midpoint NaClO concentration in A-Z transition.
4
Oligonucleotides
NaClO (mM)
4
r(CGCGCG)2
4090
880
100
8
r(CGC[m Gm]CG)
2
8
8
r(C[m Gm]C[m Gm]CG)
2
r(CGCGUGCG)/r(CGCACGCG)
5430
2360
850
8
8
r(CGCGUGCG)/r(C[m Gm]CAC[m Gm]CG)
8
8
8
8
r(C[m Gm]CGU[m Gm]CG)/r(C[m Gm]CAC[m Gm]CG)
8
To further understand the m Gm effect on the thermodynamic properties of Z-RNA, the melting
m
temperature (T ) and thermodynamic parameters were examined using CD melting experiments.
8
◦
(T ) values of m Gm-incorporated hexamer RNA increased by 6–15 C (∆T ), (Figure 2a and Table 2),
m
m
8
and octamer RNA with four m Gms in double strands containing an AU base pair increased even more,
by 15 C (Figure 2b and Table 2). Thermodynamic parameters revealed a very favorable free energy
of formation for the m Gm-incorporated Z-RNA compared to the native RNA (Table 2) suggesting
that the syn conformation of m Gm was thermodynamically favorable, and the preferred C3 endo
conformation of ribose induced by the 2 -O-methyl group also contributed to the stabilization of the
Z-form, which is consistent with the previous study [
include provision of an NMR signal from 2 -O-methyl group for NMR structure study, easy-to-prepare
phosphoramidite reagent more than 2 -O-hydroxy group for the chemical introduction of m Gm into
◦
8
8
0
0
0
1,
25]. The advantages of 2 -O-methyl substituent
0
0
8
RNA oligonucleotides, and efficient resistance to enzymatic cleavage of oligonucleotide [1,26,27].
8
Figure 2.
(a
) CD melting curves for native RNA r(CGCGCG) (back line), r(CGC[m Gm]CG)
2
2
8
8
8
8
containing one m Gm (blue line), and r(C[m Gm]C[m Gm]CG) containing two m Gms (red line).
2
(b) CD melting curves for RNA sequences containing an AU base pair, native RNA r(CGCGUGCG)/
8
8
8
r(CGCACGCG) (back line), r(CGCGUGCG)/r(C[m Gm]CAC[m Gm]CG) having two m Gms in one
8
8
8
8
8
strand (blue line), r(C[m Gm]CGU[m Gm]CG)r(C[m Gm]CAC[m Gm]CG) having four m Gms in two
strands (red line). In 5 mM sodium phosphate buffer (pH 7.0), 7 M NaClO concentrations (0.15 mM
4
base concentration).

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8
Table 2. Thermodynamic parameters for A-Z transition of various m Gm-containing RNAs.
Tm 1 ( C)
◦
∆Tm ( C)
◦
∆G298K (kcal/mol)
Oligonucleotides
r(CGCGCG)2
33.5
40.0
45.7
37.7
45.7
52.7
−
−1.1
−1.7
−2.1
−1.7
−2.5
−3.3
8
r(CGC[m Gm]CG)
+6.5
+12.2
−
2
8
8
r(C[m Gm]C[m Gm]CG)
2
r(CGCGUGCG)/r(CGCACGCG)
8
8
r(CGCGUGCG)/r(C[m Gm]CAC[m Gm]CG)
+8.0
+15.0
8
8
8
8
r(C[m Gm]CGU[m Gm]CG)/r(C[m Gm]CAC[m Gm]CG)
1
Measurement condition: [RNA] = 0.15 mM base concentration, [sodium phosphate buffer (pH 7.0)] = 5 mM,
NaClO4] = 7 M.
[
In order to obtain the structural information of Z-RNA, we performed an analysis of the
8
1
2
D NOESY spectra of r(CGC[m Gm]CG) . A complete list of H chemical shifts was shown in
2
Table S1. The NOE-restrained refinement provided an unequivocal demonstration that the structure
8
of r(CGC[m Gm]CG) is Z-RNA. We observed the strong cross-peaks in 2D-NOESY spectrum from
2
0
0
8
intranucleotide H1 /H8 NOEs of G and G and a special NOE between C1 H and 8CH of m Gm .
2
6
3
4
This indicated the syn conformation of all three guanosine residues (Figure 3b,c).
0
0
0
0
8
Figure 3.
(
a–
c
) H6/H5 of C and H8/H1 of G (8CH /H1 of m Gm) proton region of NOESY
3
8
spectra of r(CGC[m Gm]CG) in NaClO solution. The NOE connectivity pathway is shown as red
2
4
line. Intraresidue NOE cross-peaks are labeled with residue numbers. The Z-RNA structure specific
cross-peaks were observed between C amino protons and C H5 protons from the opposite strand
5
1
(
indicated as red words), as well as between C amino protons and C H5 protons from the opposite
1 5
strand (c). (d) The cross peaks of imino proton of G and amino proton of C , as well as imino proton
2
5
of G and amino proton of C , indicated Watson–Crick-type base pairs of Z-RNA. Intraresidue NOE
6
1
cross-peaks of imino and amino proton of G and G are shown.
2
6
0
0
0
0
8
The NOE connectivity path between H6/H5 of C and H8/H1 of G (8CH /H1 of m Gm )
3
4
allows for a cross-peak assignment of Z-RNA nonexchangeable resonances (Figure 3a–c). Sequential
0
0
0
assignments of C to G for the Z-form can complete the pathway: C (H6/H5 )-G (H8/H1 )-C (H6/
1
6
1
2
3
0
0
8
0
00
0
H5 )-m Gm (8CH /H1 )-C (H6/H5 )-G (H8/H1 ) (Figure 3a–c), indicating a sequence-specific
4
3
5
6
connectivity for left-handed helices.

Molecules 2018, 23, 2572
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0
The H 2 resonance was assigned based on NOESY spectra for sequential connections between
0
H6/H8 and H 2 of C and G (Figure S11). Moreover, the clear cross-peaks of the imino proton of G
(around 13.0 ppm) and the amino proton of C suggested WC base pairs (Figure 3d). The 2D NOESY of
the imino protons and other protons was used for assignment. For example, we note that the C (NH )
5
2
amino proton has cross-peaks (C (NH )/C –H5) to the C –H5 proton, and the C (NH ) amino proton
5
2
1
1
1
2
has cross-peaks to the C –H5 proton (C (NH )/C –H5) (Figure 3c). Such cross-peaks can only happen
5
1
2
5
between the two interstrand cytosines between C1 in one strand and C5 in another strand of the
Z-RNA because of their base pair stacking pattern [28,29].
We further used 31P-NMR spectroscopy to confirm these observations. We found that these
sequences as shown in Table 2 undergo a A-Z transition with increasing NaClO from 5 mM to
4
7
M (Figure 4). These results are consistent with the conformational data from the CD and proton
NMR results.
Figure 4. Monitoring of the A to Z-RNA transition by 31P NMR. 31P NMR spectra of the RNAs
at 5 mM–7 M NaClO . Green and red dots indicate the 31P peaks resulting from A-RNA and
4
Z-RNA, respectively.
8
A model of r(CGC[m Gm]CG) was constructed based on the reported Z-form structure and
2
NOE-constrained refinement [2]. The molecular dynamics simulations were performed by the standard
dynamics cascade in BIOVIA Discovery Studio 4.5 with some modifications. The conformation with
8
the lowest energy was selected as shown in Figure 5. In the m Gm-contanied Z-RNA, the C8-methyl
groups were hydrophobic and located in the outside of the helix, which is consistent with the induction
of a methyl group that strongly contributes to the increased stabilization of Z-RNA.
8
8
Figure 5. The model for r(CGC[m Gm]CG) Z-RNA structure. (
a
) m Gm is expanded in green with
the C8-methyl group, hydrogen and carbon of methyl group are coloured in yellow and purple.
) Hydrogen and carbon of methyl group are coloured in green and white. C8-methyl groups were
located outside of Z-RNA.
2
(
b

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8
Encouraged by the ability to use m Gm to stabilize Z-RNA, we study the interaction of the Z
α
8
domain and Z-RNA by using a m Gm-containing a Z-RNA and Z
α
-EGFP fusion protein, in which the
Z
α
domain is tagged with a green fluorescence protein [29]. Figure 6 indicates the visualized mode
as EGFP-mode, Cy3-mode and Merge, respectively. Lane 1, lane 2 and lane 3 in Figure 5 show free
Z-RNA labeling with fluorescent dye Cy3, a complex of Z-RNA and Z -EGFP and free Z -EGFP,
respectively. Green and red fluorescence emission visualized under EGFP-mode and Cy3-mode
distinguished Z -EGFP and Z-RNA. Because of the large molecular weight of Z -EGFP, we visualized
the complex of the Z -EGFP and Z-RNA in the upper. Lane 2 in merge mode of Figure 6 emitted
α
α
α
α
α
yellow fluorescence and clearly demonstrated the complex of Z
α
with Z-RNA. These observations
8
indicated that Z
α
-EGFP efficiently binds to the m Gm-containing Z-RNA without severe steric clashes
8
with the added methyl groups, suggesting the m Gm stabilized Z-RNA can be used to investigate the
interaction of proteins with Z-RNA.
Recent studies suggested that dsRNA pathways of RNA silencing and micro-RNA regulation
were interfered with by ADAR1 [29]. Z
α
is also reported to enhance ADAR1 editing activity on RNAs
with Z-forming sequences in vitro
similar to Z-DNA.
[29]. This suggested that Z-RNA plays important biological roles
Figure 6. Visualization of Z-RNA and Z
Lane 3: Z -EGFP only. The different modes are indicated in upper. [RNA] = 1
NaCl] = 100 mM, [Tris-HCl (pH7.0)] = 10 mM, [DTT] = 5 mM, 10% glycerol, 10
α
-EGFP protein. Lane 1: Z-RNA only, Lane 2: RNA + Z
M, [Z -EGFP] = 10
g/mL BSA. Z-RNA:
α
-EGFP,
α
µ
α
µM,
[
µ
8
8
r(CGCGUGCG)-Cy3/r(C[m Gm]CAC[m Gm]CG).
In conclusion, the results described here reveal that the newly synthesized guanosine analogue
0
2
-O-methyl-8-methyl guanosine dramatically stabilizes Z-RNA, which arises from the syn conformation
8
0
of the m Gm base and the C3 endo conformation of ribose. The oligonucleotides with AU base
pairs can convert into Z-RNA. In addition, researchers can utilize the Z-RNA stabilizer to study the
interaction of Z-RNA sequences with the Zα domain. Using the Z-RNA stabilizer, we can refine the
structure of Z-RNA based on NMR and examine the molecular basis of Z-form-specific interactions
and reactions.
3
. Materials and Methods
3
.1. Sample Preparation
We synthesized the RNA oligonucleotides by phosphoramidite chemistry with a DNA/RNA
synthesizer. We purified by RP-HPLC and prepared Z
of Oyoshi et al. [15].
α-EGFP fusion protein according to the method
3
.2. CD Experiments
We carried out CD experiments by a JASCO J-820 CD spectrophotometer (JASCO Corporation,
Tokyo, Japan). The melting curves were obtained by monitoring 285 nm. 0.3 mL samples at 0.15 mM
base concentration with 0.1–7 M NaClO , 5 mM sodium phosphate buffer (pH 7.0).
4

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3
.3. NMR Experiments
We performed NMR experiments on a Bruker AV-400 M spectrometer (Bruker, Billerica, MA, USA).
A special Micro Tube that is designed for use with reduced sample volumes was used (catalog no:
NE-H5/3-Br, New Era NMR). Dissolve RNA samples (1.0 mM) in 150 L of 90% H O/10% D O, 5 mM
µ
2
2
sodium phosphate buffer (pH 7.0), 50 mM or 3 M NaClO . 2D NOE spectra were recorded with mixing
4
times of 500 ms.
3
.4. Molecular Modeling
The model of RNA structure was manually generated based on the reported structure using the
BIOVIA Discovery Studio 4.5 (Accelrys, San Diego, CA, USA). Then molecular dynamics simulations
were performed by the standard dynamics cascade in BIOVIA Discovery Studio 4.5 with some
modifications. The structure was heating from 50 K to 300 K over 4 ps and equilibration at 300 K
with 100 ps simulation time. The save results interval in the production step was 2 ps during
1
00 ps simulation time at 300 K. 10 best conformations generated by simulation were further energy
minimized. The conformation with lowest energy was selected as shown in Figure 5.
3
.5. Elctrophoretic Mobility Shift Assay (EMSA)
We performed the experiments using RNA containing m Gm and Cy3-labeled RNA (1
with Z -GFP fusion protein (10 M) in a buffer, 10 g/mL BSA and 10% (v/v) glycerol, 100 mM NaCl,
8
µM each)
α
µ
µ
1
0 mM dithiothreitol, 10 mM Tris-HCl (pH 7.0). Nondenatured polyacrylamide gel (8%) is used and
◦
performed at 80 V and 4 C in 1× TBE buffer with 20 mM NaCl.
Supplementary Materials: The following are available online. Schemes S1 and S2: Synthetic scheme of compound
1
5
and
6
, Figures S1–S6: H-NMR spectra of synthesized compounds, Figure S7: HRMS spectra of synthesized
8
compounds, Figure S8: NOESY spectrum of m Gm, Figure S9: CD spectra of RNA shown in Table 1 at various
NaClO concentrations, Figure S10: H6/H2 of C and H8/H2 of G (8CH /H2 of m Gm) proton region of NOESY
spectra of r(CGC[m Gm]CG) , Figure S11: Monitoring of the A to Z-RNA transition by P-NMR, Table S1:
H-NMR chemical shifts δH (p.p.m. of r(CGC[m Gm]CG) Z-RNA.
0
0
0
8
4
3
8
31
2
1
8
2
Author Contributions: Y.X. designed the research. T.B., T.I., C.-D.X., and H.-L.B. carried out the experiments.
All authors analyzed the data and wrote the manuscript.
Funding: This work was supported by JSPS KAKENHI Grant Numbers 17H03091 (to Y.X.) and 16K17938 (to T.I.).
T.B. is supported by the Otsuka Toshimi Scholarship Foundation. Support from the Takeda Science Foundation is
also acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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