Characterization of Intracellular Crowding Environments with

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Characterization of intracellular crowding environments with topology-based DNA quadruplex sensors Shuntaro Takahashi, Johtaro Yamamoto, Akira Kitamura, Masataka Kinjo, and Naoki Sugimoto Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04177 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Analytical Chemistry

Characterization of intracellular crowding environments with topology-based DNA quadruplex sensors Shuntaro Takahashi,† Johtaro Yamamoto,# Akira Kitamura,§ Masataka Kinjo,§ and Naoki Sugimoto†, ‡,* †FIBER

(Frontier Institute for Biomolecular Engineering Research), Konan University, 7-1-20 MinatojimaMinamimachi, Chuo-ku, Kobe 650-0047, Japan #Biomedical

Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan §Laboratory

Japan

of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, 001-0021,

‡FIRST

(Graduate School of Frontiers of Innovative Research in Science and Technology), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan ABSTRACT: Molecular crowding creates a unique environment in cells and imposes physical constraints such as the excluded volume effect, water activity, and dielectric constant that can affect the structure and function of biomolecules. It is therefore important to develop a method for quantifying the effects of molecular crowding in cells. In this study, we developed a Förster resonance energy transfer (FRET) probe based on a guanine-quadruplex (G4) DNA motif that shows distinct FRET signals in response to crowding conditions in the presence of salt and poly(ethylene glycol). FRET efficiencies varied in different solutions, reflecting the dependence of G4 stability and topology on salt concentration and water activity. In living cells, FRET signals in the nucleus were higher than those in the cytosol; the signals in membraneless nuclear compartments (i.e., nucleolus) were especially high, suggesting that a decrease in water activity is important for the crowding effect in the nucleus. Thus, the use of DNA sensors with variable structure can elucidate the local effects of molecular crowding in cells.

The intracellular environment is occupied by a high concentration of macromolecules such as proteins, nucleic acids, and polysaccharides. The concentration of molecules in the cell is estimated to be in the range of 50– 400 mg/ml.1-2 Crowding by molecules alters the molecular environment through the excluded volume effect and changes in water activity, which in turn influence the structure and function of biomolecules. Any changes in the extent of crowding can alter the biomolecular behavior;3 for instance, the progression of amyloidosis is accelerated under crowded conditions.4 Molecular crowding also affects the stability and conformation of nucleic acid (both DNA and RNA) structures,5 leading to the formation of non-canonical DNA structures such as a guanine-quadruplex (G4) and i-motif5 that alter DNA replication and transcription and can cause cancer.6-7 Although a link between molecular crowding and cellular function has been suggested, detailed spatiotemporal information on this phenomenon is lacking. A sensing technology is required to quantitatively analyze the effects of molecular crowding in cells. Until now, technology based on Förster resonance energy transfer (FRET) has been employed that consists of

flexible and fluorescence-labeled polymers or fluorescent proteins connected to naturally disordered peptides.8-10 The flexible polymer regions become compacted according to the extent of crowding due to the excluded volume effect—one of the major factors affecting protein structure and function—resulting in a change in FRET efficiency. Other physical features of molecular crowding include a decrease in water activity and consequent changes in the dielectric constant, which can significantly affect highly charged polymers such as oligonucleotides.5, 11-13 Although proteins are difficult to design and synthesize owing to their complex three-dimensional structure, chemical synthesis of oligonucleotides that reversibly form a tertiary structure is relatively straightforward. The equilibrium reaction for the formation of the tertiary structure is predictable and tunable via sequence changes.14-16 Thus, oligonucleotides are a suitable material for analyzing various aspects of cellular function that are influenced by molecular crowding. In this study, we designed and developed oligonucleotide sensors to analyze the crowding environment in cells using FRET. We used the human

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telomere DNA sequence G3(T2AG3)3 since it can form different G4 structures depending on the molecular environment (Figure 1).5 In the presence of Na+ and K+, this sequence has anti-parallel and mixed G4 topology, respectively. Addition of a crowding reagent such as poly(ethylene glycol) (PEG) transforms the topology to a parallel structure. Thus, DNA topology presumably reflects the molecular environment. We therefore used the human telomere sequence as the sensor unit in our probe.

Figure 1. G4 structures of human telomere sequence and possible conformations of the DNA probe H-telo with the following G4 topology: (a) anti-parallel in NaCl solution, (b) mixed in KCl solution, and (c) parallel in KCl solution with concentrated PEG. For FRET analysis, Cy3 was attached to the 3' terminus of human telomere G4 (Figure S1). To the 5' terminus of human telomere G4, hairpin (HP) DNA was conjugated, and Cy5 was attached to the thymine at the third base position of the HP region (henceforth referred to as Htelo) (Figure S2). The second phosphate backbone of the HP region was replaced with thiophosphate to prevent degradation of the probe in cells. The HP region consisted of a 19-bp stem stabilized by a GAAA tetra loop.17 Different topologies of human telomere G4 exhibited variable distances between the 5' and 3' termini (Figure S1). The distance between Cy3 and Cy5 can be altered by the spatial geometry of G4 and hairpin moieties induced by topology differences in G4.18-19 Furthermore, the effect of fluorescence quenching by the G quartet can occur with various spatial geometries of the fluorophore depending on the G4 topologies. Thus, we speculated that these factors reflect changes in FRET efficiency. We

confirmed that the G4 unit of the DNA probe had antiparallel, mixed, and parallel topologies in 100 mM NaCl, 100 mM KCl, and 20 wt% PEG with an average molecular weight of 200 (PEG200), respectively, by circular dichroism analysis (Figure S3). We also determined by ultraviolet (UV) melting curve analysis that HP DNA did not unfold under the experimental conditions (Figure S4). We used the cMyc G4 unit (Figure S3)—which has parallel topology in any type of solution—and the HP without the G4 unit (henceforth referred to as cMyc and HP, respectively) as references. All sequences used in this study are shown in Table S1, and the possible conformations of DNA probes are shown in Figure 1. FRET titration experiments were conducted after annealing of the probe in the absence of any salts, after which various concentrations of salt were added to induce G4 structure formation; fluorescence spectra were then obtained at 37 °C. In the case of NaCl titration, the fluorescence spectra at 532-nm excitation showed two peaks corresponding to Cy3 (donor at 568 nm) and Cy5 (acceptor at 666 nm) (Figure 2a). Increasing the concentration of NaCl reduced and enhanced the intensity of donor and acceptor fluorescence, respectively. A similar dependence of the fluorescence on salt concentration was observed in the presence of KCl (Figure 2b). These results indicate that FRET efficiency was increased by the compaction of the G4 structure, which decreased the distance between fluorophores, and also by the increase in the fraction of H-telo forming G4 depending on Na+ and K+ concentrations. On the other hand, HP lacking a G4 structure did not show spectral changes in the presence of NaCl (Figure S5). These results provide evidence that G4 formation by H-telo can be detected as a change in FRET behavior. We performed KCl titration of H-telo in the presence of 20 wt% PEG200 and found a decrease and increase in donor and acceptor fluorescence, respectively, with increasing KCl concentration (Figure 2c). Interestingly, changes in FRET patterns for each fluorescence intensity of donor and acceptor varied with KCl titration. Thus, FRET signals from H-telo include structural information corresponding to different G4 topologies under a given crowding condition.

Figure 2. Fluorescence titration results of H-telo at 37 °C in the presence of (a) NaCl, (b) KCl, and (c) KCl with 20 wt% PEG200 excited at 532 nm. The solution was buffered with 30 mM Tris-HCl (pH 7.0). The salt concentrations were 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.3, 12.5, 25, 50, and 100 mM.


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Analytical Chemistry

Figure 3. Changes in H-telo FRET efficiency in response to (a) salt (NaCl, KCl, and KCl with 20 wt% PEG200); (b) temperature (25 °C, 37 °C, and 42 °C); and (c) crowding (PEG200, Ficoll70, and BSA). The solution was buffered with 30 mM Tris-HCl (pH 7.0), and the temperature was 37 °C unless otherwise indicated. To quantitatively determine whether the FRET signals provide information on G4 topology, we calculated FRET efficiency as an E value, which is the ratio of the fluorescence intensity of the acceptor to the sum of the donor and acceptor intensities (I666 / [I666 + I568], where I568 and I666 are the fluorescence intensities at 568 and 666 nm, respectively). Figure 3a shows the salt concentration dependency of the E value of H-telo titrated with NaCl, KCl, and KCl with 20 wt% PEG200. To determine the maximum and minimum E values (Emax and Emin), respectively, the plots were analyzed according to the binding isotherm of metal ions (see Supporting Information). Since G4 formation and stability depend on salt concentration, Emax and Emin indicate the FRET efficiencies when 100% of probes form G4 and are completely unfolded, respectively. The Emax values of Htelo were 0.48 for NaCl titration, 0.58 for KCl titration without PEG200, and 0.43 for KCl titration in the presence of 20 wt% PEG200 (Table 1). On the other hand, the Emax values of HP were nearly constant under the same conditions (0.61–0.63 in all conditions in Figure S6). Thus, each Emax value of H-telo reflected the G4 topology in a particular molecular environment. To confirm these values, we performed the same titration using cMyc, which has a parallel topology in any solution. We found that Emax in the presence of NaCl and KCl was 0.45 and 0.44, respectively (Figure S6); these were very close to the value of H-telo titrated with KCl + 20 wt% PEG200 and suggest that Emax has a parallel structure. In the tertiary structures of anti-parallel and mixed G4 topologies, the distances between the 5' and 3' termini were 2.1 and 0.87 nm, respectively (Figure S1). We expected that these would correspond to lower and higher FRET efficiencies, respectively, relative to the parallel topology (0.91 nm). However, this was not the case because G4 in the NaCl condition was not sufficiently stable to form all G4 topological types at 37 °C (Figure S4). The similar Emin values of H-telo in the NaCl and KCl titration without PEG200 indicate that the DNA structure completely unfolded in the absence of salt, while the relatively high Emin value (0.38) in the titration with KCl with 20 wt% PEG200 suggests that PEG200 facilitates G4 formation even without salt. As demonstrated above, the E value can serve as an index for evaluating the conditions of the solution

according to the stability and topology of G4. Indeed, with increasing temperature, E values shifted toward higher KCl concentration (Figure 3b). The concentrations (C1/2) when the E value was half of Emax were 5.0 (25 °C), 35 (37 °C), and 63 mM (42 °C), respectively. These results indicate that the E value changes in response to crowding factors that (de)stabilize G4. To assess the crowding environment, we used Ficoll70 and bovine serum albumin (BSA) as crowders in addition to PEG200. For each change in the E value at 37 °C (Figure 3c), the C1/2 of 20 wt% Ficoll70 was 2.4 mM, which was lower than the value obtained in the absence of crowding molecules (5.0 mM) but higher than that with 20 wt% PEG200 (0.5 mM). On the other hand, the C1/2 of 20 wt% BSA was larger (50 mM) than that of the other crowders. Emax values of Ficoll70 (0.46) and BSA (0.47) were similar to each other and close to that of PEG200 (0.48), although human telomeric G4 has a mixed topology in the presence of Ficoll70 and BSA.20-21 The difference in Emax values obtained by adding Ficoll70 and BSA as compared to KCl without a cosolute suggests a decrease in the dielectric constant under crowding conditions. Table 1. Maximum (Emax) and minimum (Emin) E values of each DNA DNA H-telo HP cMyc

Condition NaCl KCl KCl + PEG200 NaCl KCl KCl + PEG200 NaCl KCl KCl + PEG200

Emax 0.48±0.06 0.58±0.03 0.43±0.01 0.63±0.01 0.63±0.01 0.61±0.01 0.45±0.01 0.44±0.03 0.43±0.01

Emin 0.29±0.01 0.29±0.01 0.39±0.01 0.63±0.01 0.63±0.01 0.61±0.01 0.32±0.01 0.32±0.01 0.38±0.01

A phase diagram of FRET efficiency as a function of KCl and crowder concentrations was generated based on the E values (Figure 4). In the case of PEG200, high E values were observed over a broad range of KCl concentrations (> 1 mM). In contrast, Ficoll70 had little effect, while BSA lowered the E value at all KCl concentrations tested. These results suggest that physical factors including the excluded volume effect and decrease in water activity differ for each crowder, with corresponding variations in G4 topology and stability. Thus, H-telo can be used as a sensor to estimate physical properties of the solution of interest.



Figure 4. Phase diagram of E value of H-telo at 37 °C based on KCl and (a) PEG200, (b) Ficoll70, or (c) BSA concentrations. Dotted lines indicate the region of constant KCl concentration.

Figure 5. FRET analysis of living HeLa cells injected with DNA probes at 25 °C. (a) E values of the nucleus and cytosol of cells with or without gramicidin treatment. (b–d) F values of cells injected with (b) H-telo into the cytosol; (c) H-telo into the nucleus; and (d) HP into the nucleus. To investigate how sensor oligonucleotides function in cells, we introduced each probe into the nucleus and cytosol of living HeLa cells by microinjection and then quantified Cy3 and Cy5 fluorescence intensities of injected DNA probes under a confocal microscope to determine the E value of each injected area. H-telo in the nucleus had a low E value (0.31), which was comparable to or less than in vitro Emin values (Figure 5a). Similar trends in E value were observed for cMyc and HP. We also confirmed that the decrease in E values was observed in Hela nuclear fraction but not in the solution containing the G4-binding protein nucleolin (Figure S7). These results suggest that proteins that non-specifically interact with DNAs bind to sensor oligonucleotides in the intracellular conditions and drastically decrease the E values. The lower E value for cMyc could be due to nonspecific interaction between the G quartet and proteins, although these could be inhibited by the loops of H-telo (Figure 1). The decrease in E value of HP was due not only to salt concentration but also to other environmental factors. Furthermore, E values were lower in the cytosol than in the nucleus, suggesting a lower dielectric constant in the former. We examined whether perturbation of cellular environments would affect probe responses. After injecting the probes, we treated cells with 1 µg/µl gramicidin, which induces the free movement of monovalent cations into and out of cells. All E values decreased except that of H-telo in the cytosol (Figure 5a), implying that although the probes were destabilized in the nucleus due to a decrease in cations, for H-telo in the cytosol, crowding enhanced G4 formation or altered G4 topology to one with a relatively high Emax such as anti-

parallel and mixed forms, possibly in order to maintain the cation concentration in the cell. In fact, we recently reported that the KCNH1 gene encoding a K+ ion channel had clusters of G4 regions and that G4 formation was important for maintaining a high intracellular concentration of K+.7 We also scanned cellular images to examine the local environment and obtain FRET signals to calculate F value ([fluorescence of acceptor − background fluorescence] / fluorescence of donor), which differs from E value in that it emphasizes the change in FRET efficiency. Upon injection of H-telo into the cytosol of HeLa cells, the F value in the cytosol was < 0.15 (Figure 5b). Although there were some areas with a relatively high F value (~0.15), these results indicate that H-telo may not stably form a G4 structure in the cytosol. On the other hand, when Htelo was injected into the nucleus, the F value (0.2–0.35) was higher (Figure 5c), implying that H-telo formed a stable G4 structure. Interestingly, some spots in the nucleus had a high F value (0.30–0.35); these could correspond to membraneless organelles within the nucleus such as the nucleolus, which is densely packed with ribosomal RNA and thus represents a distinct molecular crowding environment. The phase diagrams in Figure 4 show that E values of PEG200 tended to increase with KCl concentrations, whereas those of BSA showed the opposite trend. Thus, at certain KCl concentrations in the cell, the molecular environments of the cytosol and nucleolus are BSA- and PEG200-like, respectively, as compared to that of the nucleus. The crowding effect may also induce a mixed topology in the nucleolus, whereas G4 had a parallel topology in other nuclear regions. As a control experiment, we injected HP into the nucleus of


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Analytical Chemistry HeLa cells and found that the F value in the nucleus was low (~0.2), especially in some regions (Figure 5d), suggesting that HP unfolding occurred at these sites. This opposite behavior of HP as compared to H-telo could reflect differences in the crowding effect on each structure. A recent study reported similar results in which DNA and RNA duplexes were shown to melt inside model membraneless organelles within the nucleus.22 Our system can explain this phenomenon from the viewpoint of molecular crowding; as previously shown, a reduction in water activity by crowding reagents decreases the stability of canonical DNA duplexes but increases that of non-canonical G4s.11 Thus, our findings suggest that the crowding environment varies according to the region within the cell. In particular, crowding conditions in the nucleus may be defined not only by the excluded volume effect but also by other factors (e.g., water activity). Since modifications and reactions related to DNA occur in the nucleus, the oligonucleotide sensor developed in this study is highly useful for analyzing the crowding environment in this organelle. In conclusion, we developed a DNA sensor that can be used to monitor the effects of molecular crowding within cells and can be applied to investigations of how this affects cellular function under normal and pathological conditions.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website; this includes materials, methods, and sequences used in this study; sequence design; and UV and FRET data (Table S1 and Figures S1–S7).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; tel.: +81 78-303-1457; fax: +81 78-303-1495.

Notes The authors declare that they have no competing financial interests.

ACKNOWLEDGMENT We thank Ms. M. Izumi for technical assistance in performing the experiments. We also thank for Dr. H. Kashida for valuable discussion. This work was supported by the Grants-in-Aid for Scientific Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems” from Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan; Japan Society for the Promotion of Science (KAKENHI grant no. JP17H06351); MEXT-Supported Program for the Strategic Research Foundation at Private Universities (2014– 2019), Japan; Hirao Taro Foundation; Chubei Itoh Foundation; and Shimadzu Science Foundation.

REFERENCES 1. Zimmerman, S. B.; Trach, S. O., Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J. Mol. Biol. 1991, 222 (3), 599-620.

2. Kuznetsova, I. M.; Turoverov, K. K.; Uversky, V. N., What macromolecular crowding can do to a protein. Int. J. Mol. Sci. 2014, 15 (12), 23090-140. 3. Rivas, G.; Minton, A. P., Toward an understanding of biochemical equilibria within living cells. Biophys.Rev. 2018, 10 (2), 241253. 4. Ambadipudi, S.; Biernat, J.; Riedel, D.; Mandelkow, E.; Zweckstetter, M., Liquid-liquid phase separation of the microtubulebinding repeats of the Alzheimer-related protein Tau. Nat. Commun. 2017, 8 (1), 275. 5. Nakano, S.; Miyoshi, D.; Sugimoto, N., Effects of molecular crowding on the structures, interactions, and functions of nucleic acids. Chem. Rev. 2014, 114 (5), 2733-58. 6. Takahashi, S.; Brazier, J. A.; Sugimoto, N., Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (36), 9605-9610. 7. Tateishi-Karimata, H.; Kawauchi, K.; Sugimoto, N., Destabilization of DNA G-Quadruplexes by Chemical Environment Changes during Tumor Progression Facilitates Transcription. J. Am. Chem. Soc. 2018, 140 (2), 642-651. 8. Gnutt, D.; Gao, M.; Brylski, O.; Heyden, M.; Ebbinghaus, S., Excluded-volume effects in living cells. Angew. Chem. Int. Ed. 2015, 54 (8), 2548-51. 9. Currie, M.; Leopold, H.; Schwarz, J.; Boersma, A. J.; Sheets, E. D.; Heikal, A. A., Fluorescence Dynamics of a FRET Probe Designed for Crowding Studies. J. Physs. Chem. B 2017, 121 (23), 5688-5698. 10. Liu, B.; Aberg, C.; van Eerden, F. J.; Marrink, S. J.; Poolman, B.; Boersma, A. J., Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding. Biophys. J. 2017, 112 (9), 19291939. 11. Miyoshi, D.; Nakamura, K.; Tateishi-Karimata, H.; Ohmichi, T.; Sugimoto, N., Hydration of Watson− Crick Base Pairs and Dehydration of Hoogsteen Base Pairs Inducing Structural Polymorphism under Molecular Crowding Conditions. J. Am. Chem. Soc. 2009, 131 (10), 3522-3531. 12. Nakano, S.; Karimata, H. T.; Kitagawa, Y.; Sugimoto, N., Facilitation of RNA enzyme activity in the molecular crowding media of cosolutes. J. Am. Chem. Soc. 2009, 131 (46), 16881-8. 13. Nakano, S.; Karimata, H.; Ohmichi, T.; Kawakami, J.; Sugimoto, N., The effect of molecular crowding with nucleotide length and cosolute structure on DNA duplex stability. J. Am. Chem. Soc. 2004, 126 (44), 14330-14331. 14. Breslauer, K. J.; Frank, R.; Blocker, H.; Marky, L. A., Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U. S. A. 1986, 83 (11), 3746-50. 15. Freier, S. M.; Kierzek, R.; Jaeger, J. A.; Sugimoto, N.; Caruthers, M. H.; Neilson, T.; Turner, D. H., Improved free-energy parameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. U. S. A. 1986, 83 (24), 9373-7. 16. Sugimoto, N.; Nakano, S.; Katoh, M.; Matsumura, A.; Nakamuta, H.; Ohmichi, T.; Yoneyama, M.; Sasaki, M., Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 1995, 34 (35), 11211-6. 17. Antao, V. P.; Lai, S. Y.; Tinoco, I., Jr., A thermodynamic study of unusually stable RNA and DNA hairpins. Nucleic Acids Res 1991, 19 (21), 5901-5. 18. Ying, L.; Green, J. J.; Li, H.; Klenerman, D.; Balasubramanian, S., Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (25), 14629-34. 19. Ray, S.; Bandaria, J. N.; Qureshi, M. H.; Yildiz, A.; Balci, H., G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (8), 29905. 20. Hansel, R.; Lohr, F.; Foldynova-Trantirkova, S.; Bamberg, E.; Trantirek, L.; Dotsch, V., The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions. Nucleic Acids Res 2011, 39 (13), 5768-75. 21. Dhakal, S.; Cui, Y.; Koirala, D.; Ghimire, C.; Kushwaha, S.; Yu, Z.; Yangyuoru, P. M.; Mao, H., Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions. Nucleic Acids Res 2013, 41 (6), 3915-23. 22. Nott, T. J.; Craggs, T. D.; Baldwin, A. J., Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. Nat. Chem. 2016, 8 (6), 569-75.




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