Letter pubs.acs.org/ac
Real-Time Monitoring of G‑Quadruplex Formation during Transcription Tamaki Endoh,† Ambadas B. Rode,† Shuntaro Takahashi,† Yuka Kataoka,‡ Masayasu Kuwahara,‡ and Naoki Sugimoto*,†,§ †
Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojima-Minamimachi, Kobe, 650-0047, Japan ‡ Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan § Graduate School of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-Minamimachi, Kobe, 650-0047, Japan S Supporting Information *
ABSTRACT: Cotranscriptional folding of an RNA transcript enables formation of metastable RNA structures. Thermodynamic and kinetic properties of RNA G-quadruplex formation have previously been investigated using purified guanine-rich oligonucleotides. Here, we describe a method for analysis of cotranscriptional dynamics of the G-quadruplex formation based on real-time monitoring of the fluorescence of Gquadruplex ligands. For RNA sequences with the potential to form mutually exclusive hairpin or G-quadruplex structures, the efficiency of G-quadruplex formation during transcription depended on position of the hairpin forming sequence. The real-time monitoring enabled evaluation of environmental effects on RNA dynamics, as we demonstrated facilitation of post-transcriptional G-quadruplex formation under molecular crowding conditions. The strategy demonstrated here provides folding insights into the G-quadruplex during transcription that should be involved in gene regulation.
T
G4 structures formed by oligonucleotides using complex experiments such as NMR and circular dichroism (CD) spectrometry, which require high concentrations of nucleic acids, or gel electrophoresis, which is time-consuming.11 Importantly, these techniques are not able to evaluate the effects of the transcriptional process on nascent G4 structures that should impact the G4 functions inside cells. In this study, we describe a technique that allowed us to monitor the cotranscriptional folding of the RNA G4s by using fluorescent probes specific for the G4 structures. N-Methyl mesoporphyrin (NMM), berberine, and thioflavin T (ThT) have higher affinity for G4 than for duplex regions of nucleic acid and emit fluorescence upon binding to the G4 structures.13 We recently synthesized a ThT analogue, in which the methyl group at the N3 position on the benzothiazole ring was replaced with a hydroxyethyl group.14 This analogue, ThT-HE, emits higher relative fluorescence intensity after binding to G4 than does the parental ThT due to lower background signal resulting from lower affinity for duplex.14 We envisioned that
he RNA G-quadruplex (G4) is noncanonical structure formed by guanine-rich transcripts.1 In cells, RNA may have a higher propensity for G4 formation than DNA due to lack of a complementary strand that can form Watson−Crick base pairs and to high stability. An RNA G4 is generally more stable than that formed by the same DNA sequence.2 It has been suggested that G4 structures in transcripts may function as regulators of various biological reactions such as alternative splicing,3,4 translation initiation,4,5 ribosomal frameshifting,6 and protein folding.7 The existence of the RNA G4 structures in cells was recently demonstrated using a G4-specific antibody.8 Nascent RNA folds during transcription in a process referred to as cotranscriptional folding.9 Folding has directionality as transcription proceeds in the 5′ to 3′ direction; therefore, sequences in the 5′ region of a transcript may form transient metastable structures. In the case of G4 formation, when cytosine and uracil are located in the 5′ flanking region of the region with quadruplex forming potential (QFP), a hairpin structure may form during cotranscriptional folding even when the G4 structure is more stable. Recent biochemical and biophysical studies suggest the importance of the alternative hairpin structures formed by the QFP sequence and its flanking regions for the function of G4 structures.10−12 Previous studies have analyzed the conformational transitions and equilibria of © XXXX American Chemical Society
Received: November 20, 2015 Accepted: January 25, 2016
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DOI: 10.1021/acs.analchem.5b04396 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
present at 5 μM concentration were much faster than the observed signal change after addition of KCl (Figure S2 of the Supporting Information). The signal change due to berberine binding was not significant in the stopped-flow experiments (data not shown). Observed binding constants between the prefolded G4 structure and the G4 ligands at 37 °C (Ka) were considerably different depending on the ligands (Figure S3 and Table S2 of the Supporting Information). On the basis of these results, the similar ktr values calculated from the fluorescence and CD signals suggest that the change of the fluorescence signal reflects the RNA conformational transition, and the kinetics are not affected by the presence of 5 μM G4 ligand irrespective of its binding affinity to the G4 structure. However, in the presence of 25 μM ligand, the ktr values were significantly larger than the value calculated from the CD experiment (Figure S4 and Table S3 of the Supporting Information); at high ligand concentration, cooperative binding of the ligands to the G4 structure might facilitate the transition.15 The amplitude of the fluorescence increase of berberine was lower than other ligands as expected from low quantum yield previously reported.16 ThT had higher background signal in the presence of LiCl than did ThT-HE, possibly caused by nonspecific interaction of ThT with hairpin.14 Thus, NMM and ThT-HE were used for further experiments. To investigate the cotranscriptional G-quadruplex formation, fluorescence signals of NMM and ThT-HE were monitored during transcription. We designed several DNA templates to evaluate the effect of hairpin location on cotranscriptional G4 formation; all have the same 23-nucleotide sequence between the T7 promoter and the HpG4 sequences of interest to ensure that the regions of interest are transcribed after a stable elongation complex has been formed (Figure 3a).17 Secondary structures and stabilities of hairpin conformers of HpG4 regions predicted by Mfold program18 are shown in Figure 3b. The HpG4-1 transcript has a sequence with potential to form a hairpin 3′ of the QFP sequence. The HpG4-2 transcript has a hairpin-forming sequence to be of similar stability to that in the HpG4-1 transcript 5′ of the QFP sequence. The mut-G4 transcript can form the hairpin structure but not the G4 structure. The fluorescence signals of 5 μM NMM and ThT-HE were monitored with transcription of the DNA templates (Figure 4a,b). During transcription of HpG4-1 and HpG4-2 sequences (0−900 s), the fluorescence signals increased with increasing the reaction time. No change in fluorescence with transcription of the mut-G4 sequence suggested that the increase of fluorescence signals reflects cotranscriptional G4 formation. After 15 min transcription reaction, DNase was added to the reaction mixture and the fluorescence signals were continuously monitored (1300−10000 s). Further increase in the fluorescence signals observed for both HpG4-1 and HpG4-2 templates suggested post-transcriptional RNA conformational transition from the metastable hairpin to the G4 structure. Smaller amplitudes of the fluorescence increase after addition of DNase, which were observed with the HpG4-1 transcript comparing to the HpG4-2, indicated more efficient G4 formation of HpG4-1 during the transcription reaction. It is considered that the HpG4-1 cotranscriptionally formed G4 structure since the region of QFP is transcribed in advance to the hairpin forming region (Figure 4c). In contrast, the HpG4-2 was expected to preferentially form the metastable hairpin structure during transcription that transitions to the G4 structure after whole region was transcribed (Figure 4d). On
cotranscriptional folding of G4 could be monitored using these fluorescent G4 ligands (Figure 1).
Figure 1. Schematic of real-time monitoring of G4 formation. (a) Principle of real-time monitoring of G4 formation during transcription by using fluorescent G4 ligands. (b) Fluorescent G4 ligands used in this study.
To validate that the fluorescent G4 ligands can detect RNA conformational transitions, we analyzed the conformational transition of an RNA oligonucleotide, HpG4-1 (Figure 2a). HpG4-1 was previously shown to form hairpin or G4 structures depending on cation concentrations.12 CD analyses indicated that the HpG4-1 oligonucleotide formed a hairpin-like structure, as shown by a negative peak at 206 nm, in the absence of KCl, and a parallel G4 structure, with a spectrum characterized by positive and negative peaks at 265 and 240 nm, respectively, in the presence of 100 mM KCl (Figure S1 of the Supporting Information) as described previously.12 An RNA conformational transition from the hairpin to G4 structure was induced by addition of 100 mM KCl at 37 °C. The observed kinetic constant for the conformational transition (ktr) was calculated to be 8.64 × 10−4 s−1 (Table 1) by fitting the CD ellipticity at 265 nm (Figure 2b) using a single exponential equation (Equation S1 in the Supporting Information). The conformational transition of HpG4-1 oligonucleotide (250 nM) was then monitored by analysis of fluorescence signals of the G4 ligands. KCl or LiCl was added at a final concentration of 100 mM, and fluorescence signals of the ligands (present at 5 μM) were continuously monitored at 37 °C (Figure 2c−f). Fluorescence signal increased as a function of time after KCl addition; no signal change was observed after addition of LiCl. The ktr values calculated from changes in the fluorescence after addition of KCl in the presence of each of the four G4 ligands were similar to that calculated from the CD experiment (Table 1). Stopped-flow analyses indicated that the interactions between G4 structure, which was prefolded in a buffer containing 100 mM KCl, and NMM, ThT, or ThT-HE B
DOI: 10.1021/acs.analchem.5b04396 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 2. Time course of the conformational transition of HpG4-1 oligonucleotide at 37 °C. (a) Hairpin and G4 structures formed by HpG4-1 oligonucleotide. (b) Time course of CD ellipticity at 265 nm of HpG4-1 oligonucleotide (5 μM) after addition of 100 mM KCl in a buffer containing 50 mM MES-LiOH (pH 7) and 0.01% Tween 20. (c−f) Normalized fluorescence intensities of G4 ligands (c) NMM, (d) berberine, (e) ThT, and (f) ThT-HE after addition of 100 mM KCl (blue) or LiCl (pink) to 250 nM HpG4-1 oligonucleotide in a buffer containing 50 mM MES-LiOH (pH 7), 250 nM HpG4-1, 0.01% Tween 20, and 5 μM ligand. Reaction buffers with NMM and berberine contained 0.1% DMSO derived from stock solutions of the ligands. Graphs show averaged signals of at least four samples.
Table 1. Observed Kinetic Constants for the Conformational Transition (ktr) of HpG4-1 Oligonucleotide in the Presence or Absence of 5 μM Ligand at 37 °C
a
ligand
NMM
berberine
ThT
ThT-HE
absencea
ktrb (10−4 s−1)
9.79 ± 0.16
8.62 ± 0.84
8.01 ± 0.30
8.11 ± 0.28
8.64 ± 0.62
Calculated from CD ellipticity. bktr values are means ± SD.
the basis of the results, the efficiency of G4 formation during transcription depends on whether the hairpin-forming sequence is 5′ or 3′ of the QFP sequence. Differences in RNA conformational dynamics in cell-free systems and in cells have been reported. 19 In vitro, thermodynamic properties of nucleic acid structures are changed by addition of cosolutes that mimic intracellular crowded conditions.20 Polyethylene glycol (PEG) destabilizes secondary structures, whereas it stabilizes G4 structures.20,21 We hypothesized that the G4 formation would be facilitated under the molecular crowding conditions. Templates were transcribed in the presence of 20 wt % PEG with an average molecular weight of 200 (PEG200), and fluorescence signals of NMM and ThT-HE were monitored (Figure 5a,b). The fluorescence increased with trends similar to those observed in dilute conditions; larger amplitude of fluorescence increase after
addition of DNase was observed with HpG4-2 than HpG4-1, and no fluorescence increase was observed upon transcription with mut-G4. HpG4-1 and HpG4-2 transcripts were purified and mixed with NMM or ThT-HE in the same buffer condition with the transcription reaction. Linear correlations of fluorescence intensities versus RNA concentrations ranging from 0 to 2000 nM were confirmed (Figure S5 of the Supporting Information). Concentrations of HpG4-1 and HpG4-2 transcripts in the reaction mixtures in the absence and presence of 20 wt % PEG200 were estimated from end point fluorescence intensities, which were obtained by fitting the fluorescence change after addition of DNase to Equation S1 (Table S4); the fitting also provides observed kinetic constants for the RNA conformational transition after transcription was terminated (ktr after transcription) (Table 2). In the presence of PEG200, both C
DOI: 10.1021/acs.analchem.5b04396 Anal. Chem. XXXX, XXX, XXX−XXX
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comparing to the dilute condition (Figure S7 of the Supporting Information). PEG200 might enhance association of RNA polymerase with DNA template.22 Concentrations of HpG4-2 transcripts tended to be less than HpG4-1 both in the presence and absence of PEG200. The sequence composition might affect the transcription efficiency through cotranscriptionally formed stable hairpin or DNA/RNA hybrid G4 structure that suppresses the transcription reaction.23,24 DNA/RNA hybrid G4 structure formed on the template DNA potentially resists DNase and affects fluorescence intensity through binding to the G4 ligands.24 However, it is expected that the signal changes observed after addition of DNase are mainly caused by conformational transition of RNA transcripts because more than 10 copies of RNA were produced from the template DNA during the 15 min transcription. Since the fluorescence signals linearly increased with RNA concentrations (Figure S5), fluorescence intensity at 15 min transcription relative to the end point intensity indicates an efficiency of G4 formation during the transcription reaction (Figure 5c,d). Both HpG4-1 and HpG4-2 significantly increased the efficiency of G4 formation in the presence of PEG200 irrespective of the G4 ligands. Stabilization of G4 structure under the molecular crowding condition20,25 possibly accounts enhancement of the cotranscriptional G4 formation. In addition, destabilization of the metastable hairpin structure under the molecular crowding condition20,21 also facilitates post-transcriptional RNA conformational transition to G4 structure as the ktr after transcription values, which should contribute the efficiency of G4 formation, were higher in the presence of
Figure 3. Design of DNA templates for transcription of HpG4 sequences. (a) Framework of DNA template. Region indicated by X encodes sequences shown in panel b. (b) Predicated secondary structures and stabilities of hairpins formed by HpG4 and mut-G4 sequences. The −ΔG° values were predicted using Mfold. Guanine tracts, which will form G-tetrads in the G4 structures, are shown in red.
HpG4-1 and HpG4-2 were transcribed more than the dilute conditions, whereas the end point fluorescence intensities were less likely due to decreased binding affinities of NMM and ThT-HE to the G4 structure (Figure S6 and Table S5 of the Supporting Information). Increase of RNA transcripts in the presence of PEG200 was also confirmed by denaturing polyacrylamide gel electrophoresis, in which the intensity of RNA transcripts was higher in the presence of PEG200
Figure 4. Real-time monitoring of cotranscriptional G4 formation. (a, b) Normalized fluorescence intensities of (a) NMM and (b) ThT-HE during (0−900 s) and after (1300−10000 s) the transcription reaction in dilute conditions. Transcription buffer with NMM contains 0.1% DMSO derived from stock solution. Signals are averages of at least four samples. White lines indicate theoretical curves fit to Equation S1. (c) Scheme of cotranscriptional G4 formation in HpG4-1 transcript. (d) Scheme of post-transcriptional G4 formation through cotranscriptionally formed metastable hairpin structure in HpG4-2 transcript. D
DOI: 10.1021/acs.analchem.5b04396 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 5. Real-time monitoring of cotranscriptional G4 formation under molecular crowding condition. (a, b) Normalized fluorescence intensities of (a) NMM and (b) ThT-HE during (0−900 s) and after (1300−10000 s) transcription reaction in the presence of 20 wt % PEG200. Transcription buffer with NMM contains 0.1% DMSO derived from stock solution. Signals are averages of at least four samples. White lines indicate theoretical curves fit to Equation S1. (c, d) Relative efficiencies of G4 formation in HpG4-1 and HpG4-2 transcripts during transcription reaction with (c) NMM and (d) ThT-HE in the absence and presence of 20 wt % PEG200. Normalized fluorescence intensities of G4 ligands at 15 min were divided by end point fluorescence intensities obtained by the theoretical curves fit to Equation S1. Asterisks indicate two-tailed P values of less than 0.01 as calculated using Student’s t-test.
hairpin and G-quadruplex. The technique demonstrated in this study will make it possible to investigate the contributions of intracellular factors on cotranscriptional and post-transcriptional folding of RNA.
Table 2. Observed Kinetic Constant for RNA Conformational Transition after Transcription (ktr after transcription) of HpG4-1 and HpG4-2
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ktr after transcriptiona (10−4 s−1) ligand NMM
ThT-HE
a
in the absence of PEG200 in the presence of 20 wt % PEG200 in the absence of PEG200 in the presence of 20 wt % PEG200
HpG4-1
HpG4-2
4.49 ± 0.33 9.63 ± 1.20
2.65 ± 0.02 6.26 ± 0.11
5.21 ± 0.70 31.5 ± 9.47
3.51 ± 0.06 7.04 ± 0.22
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04396. Supporting materials and methods, CD spectra of HpG41 oligonucleotide, stopped-flow analyses of fluorescent G4 ligands, binding analyses between HpG4-1 oligonucleotide and G4 ligands, time course of RNA conformational transition with 25 μM ligands, fluorescence analyses using RNA transcripts, and denaturing polyacrylamide gel electrophoreses of RNA transcripts (PDF)
Values are means ± SD obtained from at least four samples.
PEG200 comparing to the dilute conditions (Table 2). The molecular crowding condition has more impact on the sequence, which has hairpin forming sequence upstream of the QFP, as the sequence preferentially forms metastable hairpin structure that post-transcriptionally transitions to the G4 structure. In conclusion, fluorometric detection of G4 formation using G4 ligands enables real-time analysis of RNA folding during transcription. Efficiency of G4 formation depended on both the relative locations of hairpin-forming and G-quadruplex-forming sequences within the transcript and on solution conditions. The location affected the efficiency of cotranscriptional folding, and the molecular crowding facilitated the post-transcriptional RNA conformational transition. Helicases, RNA chaperones, and G4 binding proteins26 and intermolecular interactions between nucleic acid strands might also alter the dynamics between the
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest. E
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Analytical Chemistry
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ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research and MEXT (Japan)-Supported Program for the Strategic Research Foundation at Private Universities (2014−2019) and The Hirao Taro Foundation of KONAN GAKUEN for Academic Research. We thank Yukiyo Tada, Ken-ichi Hase, and Misa Kinoshita for their help with the experiments.
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DOI: 10.1021/acs.analchem.5b04396 Anal. Chem. XXXX, XXX, XXX−XXX
