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. Author manuscript; available in PMC: 2023 Jun 15.
Published in final edited form as: J Am Chem Soc. 2022 May 31;144(23):10386–10395. doi: 10.1021/jacs.2c02222

Formation of vs. Recruitment to RNA-Rich Condensates: Controlling Effects Exerted by Peptide Side Chain Identity

Jiani Niu 1, Cindy Qiu 2, Nicholas L Abbott 2, Samuel H Gellman 1
PMCID: PMC9746169  NIHMSID: NIHMS1853630  PMID: 35639776

Abstract

Liquid-liquid phase separation (LLPS), which is the spontaneous formation of contiguous liquid phases with distinct compositions, has been long known in chemical systems and more recently recognized as a ubiquitous feature of cell biology. We describe a system involving biologically relevant components, synthetic peptides and total yeast RNA, that has enabled us to explore factors that underlie phase separation. Coulombic complementarity between a cationic peptide and anionic RNA is necessary but not sufficient for formation of a condensed phase in our system. In addition to a net positive charge, the peptide must present the proper type of cationic moiety. Guanidinium groups, as found in the Arg side chain, support phase separation, but ammonium groups, as found in the Lys side chain, or dimethylguanidinium groups, as found in post-translationally modified Arg side chains, do not support phase separation in our system. However, the cationic groups that do not support phase separation via interaction with RNA can nevertheless enable recruitment to a condensed phase, which reveals that the network of forces governing condensed phase formation can differ from the network of forces governing recruitment to such a phase. We introduce a new method for measuring the concentrations of components in condensed phases based on fluorine-containing additives and 19F NMR.

INTRODUCTION

Liquid-liquid phase separation (LLPS) mediated by biopolymers generates membraneless organelles and other types of condensates that are important features of the dynamic internal environment in cells.1–8 New participants in condensate formation and new physiological roles for LLPS are being uncovered at a rapid pace. Biomolecule-based phase separation phenomena can be roughly divided into two classes.9 One type of phase separation (designated class I here) relies on multivalent protein-protein or protein-nucleic acids interactions involving well-folded and specific recognition modules that are linked by flexible and often disordered segments.10–16 The other type (designated class II here) is mediated by small molecular features, such as individual protein side chains, that are dispersed within biopolymer components.17–20 Proteins or protein domains that support class II phase separation are typically disordered.17

The network of intermolecular forces that results in class II phase separation is not always clear. Such systems provide opportunities to discover new principles of molecular assembly that underlie formation of condensed phases, including but not limited to principles that have been harnessed by evolution. For example, complex coacervation resulting from interactions of polyanions and polycations is strongly influenced not only by net charge on the individual components but also by the distribution of charged groups along the polymer chains.21 The stereochemistry of peptide-based polycations or polyanions can affect the physical properties of their assemblies, with heterochiral peptides favoring liquid assemblies relative to homochiral peptides.22 The nature of charge-bearing groups can modulate the physical properties of condensed phases formed by polycation-polyanion pairing, as illustrated by variations between lysine-based and arginine-based polypeptides (ammonium vs. guanidinium).23,24

The biological roles of LLPS are not fully understood,25,26 but some functions are coming into focus. For example, P-bodies promote RNA processing in the cytoplasm by concentrating the necessary enzymes and their substrates, and the nucleolus is important for ribosome assembly.27 In addition, membraneless organelles appear to provide spatiotemporal control of cellular signaling.28 Such functions require that specific biomolecules be recruited to condensed phases within cells. Here we ask whether the molecular features necessary for formation of a biopolymer-rich condensate via class II phase separation can be distinguished from features that allow recruitment to a biopolymer-rich condensate.

RESULTS

Arginine-rich peptides derived from FUS mediate coacervation in combination with RNA.

As a prelude to addressing the formation vs. recruitment question, we sought to identify a biomimetic class II phase separation system in which a polypeptide component could be accessed by chemical synthesis. This goal was motivated by our desire to transcend the limits on amino acid composition that are imposed by ribosomal synthesis. Our exploratory efforts were based on Fused in Sarcoma (FUS), an RNA-binding protein that mediates condensate formation in vivo and that has been extensively studied in vitro.17,29–32 The FUS C-terminal domain (CTD; Figure 1a) contains three Arg/Gly-rich (RGG) motifs; similar segments occur in other RNA-binding proteins that support membraneless organelle formation.33 Evaluation of CTD fragments of varying lengths led us to 1, a 27-mer derived from FUS (471–497) (Figure 1b, S1) that supports robust phase separation when combined with RNA.

Figure 1.

Figure 1.

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(a) Schematic of the protein FUS (1–526). LCD, low complexity domain; CTD, C-terminal domain. Peptides used in this study were derived from FUS (471–497) within the Arg/Gly-rich CTD. (b) Primary sequences of peptides 1 - 5. Sites of variation are indicated by a red X, and the specific amino acid residues are shown at the bottom.

Peptide 1 contains eight native Arg residues but differs from the corresponding FUS segment in that the three natural Asp residues were replaced by Glu, to avoid aspartimide formation during peptide synthesis. Peptide 1 should have a net charge of +5 near neutral pH. Phase separation could be induced by combining 1 with total yeast RNA. Use of this highly heterogeneous RNA source was expected to minimize the possibility that formation of a condensed phase would be driven by specific RNA-peptide recognition.23,34

Formation of droplets was observed via microscopy when 0.025–0.1 mM 1 in Tris buffer, pH 7.5, was mixed with 0.25 μg/μL total yeast RNA (referred to as “RNA” below; ~0.8 mM on a per-nucleotide basis). To enable fluorescence microscopy, we included 4 mol% of a derivative of 1 bearing a fluorescein moiety (Fl-1, Figure S2). Liquid droplets could be observed via differential interference contrast (DIC), fluorescence and Z-stack modes (Figure 2a, S3a). The droplets were round at ambient temperature (i.e., without thermal annealing). Droplets could be observed to fuse upon contact with one another (Figure S3b), and droplets spread spontaneously when they contacted a surface (Figure S3c). Fluorescence recovery after photobleaching (FRAP) measurements revealed a very rapid return to uniform fluorescence after focused irradiation (within ~0.3 s; Figure 2b). Collectively, these observations indicate that the droplets represent a liquid state in which the peptide component is highly dynamic.25 It is possible that the RNA component in these droplets is less dynamic.23

Figure 2.

Figure 2.

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Arg-rich peptide 1 undergoes phase separation when mixed with 0.25 μg/μL total yeast RNA in pH 7.5 buffer. (a) Top: droplets formed by RNA and 1 at different peptide concentrations viewed via differential interference contrast (DIC). Bottom: corresponding fluorescence images in the 488 nm channel (fluorescein, Fl; attached to the peptide), false colored to green. Scale bar = 10 μm. (b) FRAP experiment. Left: A region within a droplet is photobleached (dark; diameter = 1 μm) at T = 0; right: near 100% fluorescence recovery is observed very rapidly (right, at T = 0.33 sec). Scale bar = 1 μm.

An increase in average droplet size 30 min after mixing 1+RNA was observed when the peptide concentration was increased from 0.025 mM to 0.1 mM while holding RNA concentration constant. (Droplet size varied as a function of time, because droplets slowly fused with one another.) This trend was evident from micrographs (Figure 2a) and confirmed via dynamic light scattering measurements (Figure S3d). A concomitant concentration-dependent increase in turbidity was observed over this peptide concentration range (Figure S3e).

To test the hypothesis that the condensed phase formed by combining peptide 1 and RNA corresponds to class II phase separation, i.e., formation of condensates mediated by small motifs such as side chains, rather than interaction of 1 with a specific, folded RNA partner (e.g., a G-quadruplex34), we evaluated the enantiomer of 1 (ent-1) and a “scrambled” sequence isomer of 1 (mix-1) (Figure S4). For mix-1, we changed the RGG local sequence motifs to GGR without disturbing the overall charge distribution, which can be important for phase separation.21 Formation of droplets was observed when either of these peptides was combined with RNA (Figure S4). These results suggest that the interactions between RNA and 1, ent-1 or mix-1 that result in formation of condensates do not require any particular configuration (i.e., L vs. D) or local sequence (e.g., RGG) within the peptide. Instead, phase separation seems to result from transient noncovalent interactions that involve individual side chains or other small fragments, as expected for class II phase separation. Analysis of droplet size by micros-copy, however, indicated a statistically different distribution of droplet sizes between the phase separation samples formed after 30 min by 1+RNA vs. ent-1+RNA (Figure S4).

Phase separation caused by mixing of polycations with polyanions, such Arg- or Lys-rich polypeptides and RNA, is well-known.19,24,35,36 Complex coacervation by polyions with complementary charges has been attributed partially or entirely to Coulombic attractions.35–37 Polycation-polyanion association leads to entropically favorable release of counterions that were localized around each polyion.38–41 A role for polycation-polyanion attraction in the phase behavior of 1+RNA is suggested by the observation that addition of ≥125 mM NaCl or KCl diminished the turbidity measured for 0.1 mM 1 + 0.5 μg/μL RNA in pH 7.5 buffer (Figure S5). This behavior presumably reflects the screening of polycation-polyanion association by sodium, potassium and/or chloride ions.

Peptide Side Chain Identity is Critical for Formation of a Condensed Phase.

Peptide 2, an analogue of 1 in which six of the eight Arg residues are replaced with Lys, did not support phase separation under the conditions described above. Specifically, mixing 0.1 mM Lys-rich 2 with 0.25 μg/μL RNA in pH 7.5 buffer did not induce phase separation (Figure 3), even though Arg-rich 1 and Lys-rich 2 are expected to have a similar net charge in this buffer. Thus, the different behaviors of 1 and 2 suggest that Coulombic attractions cannot fully explain phase separation in solutions of 1+RNA. Arg vs. Lys differences in complex coacervation involving proteins or synthetic polypeptides have been previously noted,17,23,24,35,42 although explanations for these observations have varied, and not all systems display such differences.30

Figure 3.

Figure 3.

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Detection of phase separation for different peptides (0.1 mM) mixed with 0.25 μg/μL RNA by turbidity measurements after 30 min incubation. Only peptides containing L-Arg, D-Arg or MMA (1, ent-1, and 3) showed significant turbidity when incubated with RNA in 50 mM Tris, pH 7.5. Data presented as means ± SD (n = 6). P values were calculated by one way ANOVA.

Peptides 3-5 (Figure 1b) are analogues of 1 that contain common post-translational modifications at six of the eight Arg positions; this peptide set allowed us to evaluate the impact of each modification on phase behavior. Formation of monomethyl-arginine (MMA; peptide 3), asymmetric dimethyl-arginine (ADMA; peptide 4) and citrulline (Cit; peptide 5) are common enzymatic post-translational modifications of arginine in vivo.43,44 MMA and ADMA retain the positive charge of Arg near neutral pH, but Cit is uncharged. We initially probed for condensed phase formation by monitoring light scattering at 340 and 600 nm (Figure 3 and S6) when each peptide (0.1 mM) was mixed with 0.25 μg/μL RNA in Tris buffer. MMA-rich peptide 3 caused a modest increase in OD340, roughly 60% of the increase observed for unmethylated peptide 1. In contrast, no phase separation was observed for ADMA-rich peptide 4 or Cit-rich peptide 5. The observations with 4 are consistent with reports that the ADMA post-translational modification impairs the ability of FUS to mediate phase separation.30 The behavior of 5 is consistent with the hypothesis that complex coacervation upon mixing 1 and RNA is driven at least in part by Coulombic attraction and requires a net positive charge on the peptide. The lack of phase separation observed with asymmetric dimethylated Arg (1 → 4), however, supports our conclusion that phase separation induced by mixing 1 and RNA cannot be fully explained by Coulombic interactions.

The Ability to Form a Condensed Phase and the Ability to be Recruited to a Condensed Phase Are Distinct Properties.

Our observation that the nature of the cationic side chain group determines whether phase separation occurs in the peptide-RNA mixtures described above allowed us to ask whether there is a distinction between the ability to support formation of a biopolymer-rich phase and the ability to be recruited to such a phase. This question was first addressed qualitatively by mixing RNA (final concentration 0.25 μg/μL) with two peptide samples (Figure 4). Peptide 1 (0.1 mM; containing 4 mol% Fl-1) was a component of each mixture to ensure formation of a condensed phase. The second peptide (0.1 mM) was varied but in each case contained 4 mol% of the derivative bearing a tetramethylrhodamine (TMR) label. Fluorescence microscopy allowed independent monitoring of the presence of the two peptides within droplets. As expected, when the second peptide was 1 or ent-1, the Fl and TMR fluorescence signals co-localized in the droplets (Figure 4a, b).

Figure 4.

Figure 4.

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Selective recruitment of peptides to RNA-rich droplets. (a-d) Colocalization of various of peptides (1, ent-1, 2, or 4 containing 4 mol% TMR-labeled derivative) with peptide 1 (containing 4 mol% Fl-labeled derivative) in the condensed phase with RNA. (e) Cit-rich peptide 5 was not detected in the droplets. 0.1 mM of each peptide and 0.25 μg/μL RNA were incubated at room temperature for 30 min before images were taken. Scale bar = 5 μm.

To our surprise, Lys-rich 2 and ADMA-rich 4 were each concentrated in droplets formed by the interaction of Arg-rich 1 and RNA (Figure 4c, d). In contrast, Cit-rich 5 was not concentrated in the droplets (Figure 4e). Selective recruitment of the cationic peptides (1, 2 and 4) but not Cit-rich 5 was observed also at lower peptide concentrations (0.025 or 0.05 mM; Figure S7). Z-stack images established that the droplets were homogeneous (Figure S8), i.e., that the Fl and TMR signals were evenly distributed within the droplets. Overall, these results indicate that the Lys-rich peptide 2 and the ADMA-rich peptide 4 can be recruited to an RNA-rich condensed phase, even though neither 2 nor 4 independently supports phase-separation with RNA under these conditions (Figure 3).

To gain further insight on formation of and recruitment to condensed phases described above, we sought to quantify the components within these phases (Figure S9a). Measuring concentrations of components within liquid droplets is challenging. Strategies based on fluorescence microscopy require complex analysis and careful controls.18,45–48 We developed a new and simple method to assess peptide concentrations in the RNA-rich condensed phases described here.

Direct measurement of condensed phase volume is difficult or impossible at the experimental scales typical for these studies, which hampers determination of component concentrations.18 We reasoned that including a fluorine-containing compound that has no preference between the condensed and dilute phases, i.e., a species that is neither concentrated in nor excluded from the condensed phase, would allow us to determine the volume of the condensed phase based on 19F NMR measurements (Figure S9b). Phase separation was induced by combining 0.1 mM 1 and 0.25 μg/μL RNA in 0.6 mL of 50 mM Tris, pH 7.5, with 5 mM of a fluorine-containing additive (identified below). After 30 min, centrifugation was used to isolate the condensed phase, which was then dissolved in 0.6 mL of 0.5 M aqueous NaCl (Figure S9c). At this stage, the concentration of the fluorine-containing compound could be determined via integration of the 19F NMR signal, based on an externally generated calibration curve. To test the assumption that the fluorine-containing additive had no phase preference, we conducted parallel studies with two simple salts (NaF and NaBF4) and three small molecules trifluoroacetyl glycine (TFA-Gly), trifluoroacetamide and N-trifluoroacetyl-D-glucosamine (F3-GlcNAc) (Figure 5). All five provided similar estimates of condensed phase volume, ~1% of the total coacervate sample (Table S1). Because physical properties vary among these five fluorine-containing compounds, the similar outcomes support our hypothesis that each of these fluorine-containing compounds is neither concentrated in nor excluded from the condensed phase.

Figure 5.

Figure 5.

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Chemical structures of five fluorine-containing probes with diverse physical properties for parallel concentration determinations via 19F NMR integration.

We used the 19F NMR-based quantification method to examine the effect on condensed phase composition of varying the concentration of peptide 1 when RNA was held constant at 0.25 μg/μL in 50 mM Tris, pH 7.5, with 5 mM NaF as the additive. As shown in Figure 6a, peptide concentration in the condensed phase increased steadily as the total peptide concentration was increased from 0.05 to 0.2 mM, but this trend diminished between 0.2 and 0.4 mM. The percentage of peptide 1 in the condensed phase was reasonably consistent (75–85%) as the total peptide concentration was varied, but a small decline was evident at the highest concentration. The condensed phase volume did not change significantly across this peptide concentration range (Figure S10b). The proportion of RNA in the condensed phase rose steadily between 0.05 and 0.2 mM peptide 1 and leveled off between 0.2 and 0.4 mM of 1 (Figure 6b). At 0.2 mM 1, the polycation:polyanion charge ratio (peptide:RNA) in the condensed phase should be ~1:1, if we assume that all basic and acidic side chains groups on the peptide (8 Arg and 3 Glu) are ionized. The predicted polycation:polyanion ratio increased to ~2:1 at 0.4 mM 1. Trends in predicted concentrations of charged groups from polyions (Figure 6c) and polyion charge ratios (Figure 6d) suggest that the condensed phase can attract an excess of the polycationic peptide, on a charge basis, relative to polyanionic RNA. Such “overcharging” within complex coacervates has been explained based on the favorable entropy associated with counterion release and a favorable combinatorial entropy arising from polycation-polyanion association modes.49,50

Figure 6.

Figure 6.

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Quantitative analysis of condensed phase components, for phase separation induced by mixing 1 with 0.25 μg/μL RNA in 50 mM Tris, pH 7.5, based on 19F NMR. Condensed phase volume at each peptide concentration was measured via 19F NMR with NaF as the reference compound as described in the text. (a) Relative distribution of peptide 1 (%) in the condensed phase (upward bars) and in the dilute phase (downward bars) at different total peptide concentrations. The estimated peptide concentration in each phase is indicated next to each bar. (b) Relative distribution of RNA (%) in the condensed phase (upward bars) and in the dilute phase (downward bars) at different total peptide concentrations. The estimated RNA concentration (per-nucleotide basis) is indicated next to each bar. (c) Estimated ionic group (Arg+ on peptide and PO4− on RNA) concentrations in the condensed phase at various peptide concentrations. (d) Charge ratio (left axis: ratio of Arg+ on peptide to PO4− on RNA; right axis: ratio of total peptide charge to PO4− on RNA) at different concentrations of peptide 1 in the condensed phase. Data presented as means ± SD (n = 4).

Peptide concentrations in RNA-rich phases containing two peptides were assessed with the 19F NMR-based method (Figure 7). Each sample contained 0.25 μg/μL RNA and 0.1 mM 1 (containing 20 mol % Fl-1) to ensure phase separation. Samples varied in the identity of the second peptide, also at 0.1 mM. The second peptide contained 20 mol % of the TMR-labeled derivative. This proportion of labeled peptide was necessary to ensure adequate sensitivity in optical measurements of concentration. Control studies showed that varying the proportion of fluorescently labeled peptide did not affect our conclusions (Figure S11a). When the second peptide sample was Arg-rich 1 or ent-1, after 30-minute incubation we detected ~90-fold enrichment (based on TMR) in the condensed phase relative to the total peptide concentration. This observation suggests that the concentration of ent-1 in the condensed phase was ~ 9 mM (Figure 7b). Citrulline-containing peptide 5 was not significantly enriched in the droplets (Figure 7b).

Figure 7.

Figure 7.

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(a) Sample preparation for the two-peptide system concentration measurements. 0.1 mM 1 (with 20 mol% Fl-1) and 0.1 mM second peptide (with 20 mol% TMR-labeling) were mixed with 0.25 μg/μL RNA in Tris buffer and incubated at room temperature for 30 min. The coacervate sample was then spun down to collect the supernatant. The concentration of each component in the supernatant was determined by UV-spectroscopy, and this information along with the volume of the condensed phase determined by 19F NMR were used to determine the enrichment index. (b) Estimated enrichment index for the TMR-labeled peptide in the two-peptide experiments. The enrichment index is the ratio of the concentration of the peptide in the condensed phase to the total concentration of that peptide in the sample. Estimated peptide concentrations in the condensed phase are shown on the right vertical axis. Tetramethylrhodamine (TMR) at 0.02 mM was used as a non-peptide control. (c) Displacement of the original peptide, 1, by the second peptide, as judged based on Fl fluorescence. Positive scale represents % of 1 remaining in the condensed phase; negative axis represents % of 1 displaced into the dilute phase. (d) Distribution of RNA (%) in condensed phase (positive scale) vs. RNA (%) in dilute phase (negative scale) when different peptides are added. Results shown here are averaged over the five fluorine-containing reference compounds shown in Figure 5. Data presented as means ± SD (n ≥ 4). P values (P > 0.1234 (ns), P < 0.0001 (****)) were calculated by one way ANOVA.

Although neither Lys-rich 2 or ADMA-rich 4 could support formation of a condensed phase with RNA under these conditions (Figure 3), each of these peptides showed ~70-fold enrichment in the condensed phase formed by 1+RNA (i.e., the concentration of 2 or 4 within droplets was ~7 mM) (Figure 7b). These results are consistent with the qualitative microscopy findings discussed above (Figure 4) and strengthen the conclusion that the ability to support formation of an RNA-rich condensed phase and the ability to be recruited to and concentrated in such a phase are distinct properties. Our data show that these properties can vary among peptides that differ in the nature of their cationic side chains but have the same net charge and the same distribution of charged groups.

Previous studies with poly-Arg and poly-Lys showed that one polymer could displace the other from complex coacervates under some conditions.24 We therefore asked whether the “scaffold” peptide 1 (with 20 mol% Fl-1) was displaced from the condensed phase by the second peptide under the conditions we employed. Upon the addition of 0.1 mM 1 or ent-1 to a pre-formed condensed phase, only a small portion (~10%) of 1 was displaced after 30 minutes, as judged by the Fl signal (Figure 7c). The displacement of 1 by 0.1 mM Lys-rich 2, ADMA-rich 4 or Cit-rich 5 was insignificant. None of the added peptides caused a significant change in the proportion of RNA in the condensed phase (Figure 7d).

Dynamics of Condensed Phase Droplets: Distinct Entry Modes.

As a complement to the studies described above, in which both peptide samples were simultaneously mixed with the RNA component (Figure S12a), we conducted experiments in which a phase-separated sample was generated by mixing 0.25 μg/μL RNA with 0.1 mM 1 (4 mol% Fl label), and the second peptide (4 mol% TMR label) was added after 2 hours (Figure S12b). Time-lapse microscopy suggested different mechanisms for entry of the second peptide into preformed droplets as a function of side chain. Upon addition of Lys-rich 2 or ADMA-rich 4, neither of which independently supports phase separation with RNA under these conditions, TMR fluorescence entered preformed droplets uniformly around the periphery and moved to the droplet center (Figure 8a). This process appeared to be complete across the sample within ~90 seconds.

Figure 8.

Figure 8.

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Time-lapse images of peptide recruitment to condensed phase droplets. (a) Peptides that did not support formation of condensed phase (2 and 4) diffused into the pre-formed droplets uniformly from the periphery. (b) Peptides that supported formation of condensed (1 and ent-1) entered the pre-formed droplets through a “patch”. Scale bar = 10 μm.

In contrast, upon addition of Arg-rich 1 or ent-1, each of which supports formation of condensed phase, TMR fluorescence first appeared in one small region of the droplet edge and then migrated across the entire droplet (Figure 8b). Some videos showed the sudden appearance of a droplet that displayed high and uniform TMR fluorescence (i.e., fluorescence detected in only the TMR channel) (Video S1). In these cases, subsequent micrographs suggested that TMR-labeled peptide from such droplets rapidly moved to the nearest edges of neighboring droplets, which initially manifested fluorescence in only the fluorescein channel. These images suggest that new droplets may have formed from the added peptide and residual RNA in the dilute phase (Figure 6b and 7d), with subsequent exchange of Fl- and TMR-labeled peptides between neighboring droplets. We could not discern whether newly formed TMR-rich droplets were associated with all cases of entry into preformed droplets.

Collectively, the different behaviors observed for Lys-rich 2 or ADMA-rich 4 vs. Arg-rich 1 or ent-1 raise the possibility that different proteins may be recruited to RNA-rich membraneless organelles via distinct mechanisms depending on their complement of basic side chains and/or their degree of post-translational modification. Such mechanistic distinctions might be correlated with different consequences in terms of cellular physiology.

DISCUSSION

The studies reported here were motivated by the widespread occurrence of liquid-liquid phase separation in cells and by enduring uncertainties about the physicochemical factors that control such phenomena and the functional roles of phase separation in biology.1–23, 25 We identified a system in which phase separation can be induced by combining an Arg-rich synthetic peptide derived from the protein FUS and heterogeneous RNA. This form of complex coacervation appears to be mediated by interactions of small moieties, such as individual side chains, that are dispersed along the flexible peptide backbone. Our system allowed us to probe the role of amino acid side chain identity in phase separation arising from polycation-polyanion interaction.

Our results show that Coulombic complementarity is necessary for peptide-RNA complex coacervation, as would be expected, but that Coulombic attraction is not sufficient for phase separation in this system. The identity of the cationic group is a critical determinant. Thus, replacing Arg residues (guanidinium groups) with either Lys or ADMA residues (ammonium or asymmetric dimethylguanidinium groups) prevents phase separation with RNA under our conditions. These observations are correlated with results reported for FUS protein self-association: Arg-to-Lys mutations in the FUS C-terminal domain diminish phase separation propensity, as does post-translational modification of Arg residues to ADMA.17,30 However, the mechanistic origins of these effects are unclear. For example, it has been proposed that diminished phase separation observed upon Arg-to-ADMA modification results from diminished cation-π attraction between dimethylguanidinium and aromatic groups in the protein relative to cation-π attraction between guanidinium and aromatic groups.30 This hypothesis seems to be inconsistent with elegant model studies showing that the ADMA side chain forms more favorable cation-π interactions with a Trp side chain than does the unmodified Arg side chain.51

The trend we observe among peptides containing 1, 3 and 4 (Figure 3), which indicates decreasing propensity to phase separate as the side chain is varied from Arg to MMA to ADMA, raises the possibility that the hydrogen bonding properties of the peptide side chains may be critical for phase-separation mediated by Arg-rich peptides or proteins and RNA. Guanidinium and phosphate groups can associate via monodentate and bidentate hydrogen bonding motifs (Figure 9a,b).52–54 The Arg side chain offers five possibilities for the monodentate H-bond motif, MMA offers four, and ADMA offers three. For the bidentate H-bond motif, the Arg side chain offers two possibilities, MMA offers one, and the ADMA side chain does not allow this motif (Figure 9c–e; the position of the Arg side chain δ carbon in Figure 9a, b relative to the methyl group(s) is constrained by the need to avoid a syn-pentane-like interaction). Thus, post-translational arginine methylation reduces H-bond options with phosphate groups on RNA, a trend that could explain the variations we observe in the abilities of peptides 1, 3 and 4 to support phase separation with RNA. A Lys ammonium group can in principle form a bidentate H-bond interaction with phosphate groups, but a computational study concluded that this interaction is less favorable than a bidentate Arg guanidinium-phosphate interaction.55 An experimental comparison of Arg oligomers vs. Lys oligomers as binding partners for RNA or DNA led to the conclusion that the hydrogen bonding of guanidinium groups to backbone phosphates could explain the higher affinity of Arg oligomers.41 Our comparisons suggest that the bidentate H-bond motif might be important to support the phase separation we observe.

Figure 9.

Figure 9.

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Possible H-bonding patterns between arginine side chains and RNA backbone phosphates. (a) Monodentate H-bond motif between an arginine side chain and a phosphate group on RNA. (b) Bidentate H-bonding motif. (c) Five monodentate (blue circles) and two bidentate (arrows) H-bonding sites possible for arginine. (d) Three monodentate (blue circles) and zero bidentate H-bonding sites possible for asymmetric dimethylated arginine. (e) Four monodentate (blue circles) and one bidentate (arrows) H-bonding sites possible for monomethylated arginine.

CONCLUSION

The studies reported here demonstrate a difference between the network of noncovalent forces between a cationic polypeptide and RNA that is required to support formation of a condensed phase and the network required for recruitment to a pre-existing condensed phase. Recruitment, at least in our system, may depend only on Coulombic attraction between polyions, presumably with concomitant counterion release, while formation of the condensed phase seems to require additional noncovalent attraction, possibly including a specific bidentate H-bonding motif (Figure 9b). The distinction between formation of and recruitment to membraneless organelles and other intracellular assemblies may prove important for elucidating the biological functions of these assemblies.

An important feature of this study is the new strategy we have developed for measuring concentrations of molecules within condensed phases. The 19F NMR-based method allowed us to determine that cationic peptides incapable of supporting phase separation in combination with RNA could nevertheless achieve relatively high concentrations (~7 mM) within a condensed phase scaffolded by an Arg-rich peptide and RNA, approaching the concentration of the Arg-rich peptide (~9 mM). The difficulty of quantifying component concentrations in condensed phases via fluorescence measurements have been discussed.47 For in vitro studies, the molecules required to induce phase separation are often not readily available in large quantities (e.g., the synthetic peptides we employed, or an expressed protein). At typical experimental scales, the condensed phase generated in such systems has a volume of ≤ 5 μL, which is difficult to measure directly with accuracy. The use of non-interactive fluorine-containing compounds, which are available in diverse forms, enables reliable and convenient concentration determinations for many parallel condensed phase samples, which is necessary for comparative studies of the type reported here. In more complicated systems, some components cannot be readily labeled with a fluorophore to enable microscopic analysis. The 19F NMR-based method introduced here can be applied to non-fluorescent components in complex coacervates, such as RNA in our case.

While our manuscript was under review, a paper appeared describing the use of 2,2,2-trifluoroethanol (TFE) as a probe to study phase separation induced by addition of YCl3 to concentrated solutions of bovine serum albumin (BSA) via 19F NMR.56 This use of a fluorine-containing additive is distinct from the method we describe here. The main goal of the studies in ref. 56 was to characterize the kinetics of phase separation. The authors used 19F chemical shift data to measure BSA concentration in the condensed phase; these measurements required an external calibration curve constructed with samples containing independently determined BSA concentrations. The effect of BSA concentration on 19F chemical shift was attributed to interactions between TFE and the protein surface. Thus, use of this method to measure macromolecule concentration in a different type of condensed phase would presumably require a new calibration curve. In contrast, our method relies on integration of 19F NMR signals after the condensed phase has been dissolved and diluted; therefore, the identity of the macromolecules that enable phase separation does not affect our measurements. Our method can be readily applied to systems with complex and diverse compositions, as illustrated above.

Although many questions remain open regarding mechanisms of phase separation in cells and the functional outcomes of such processes, it is clear that this unique mode of compartmentalization has been harnessed by evolution.2,8,25,36 Insights gained from the rapidly expanding appreciation of LLPS in biology are inspiring the exploration of new applications of LLPS in non-biological contexts.18,21,24,45 Elucidation of the principles that control the formation of condensed liquid phases and the transit of molecules between contiguous condensed and dilute phases is necessary to understand the roles of condensates in biology and to realize the potential of LLPS-based engineering in chemical systems. The insights and tools described here contribute to this long-term goal.

Supplementary Material

Supporting Information
Supporting Information AVI File
Download video file (2.8MB, avi)

ACKNOWLEDGMENT

This work was supported in part by a grant from NIGMS (R01 GM061238) and NSF (DMR-2003807).

Footnotes

The authors declare no competing interests.

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Materials (Section I), method descriptions (Section II), additional figures and tables (Section III), 19F-NMR data (Section IV) and characterization data for all synthesized peptides (Section V) (PDF).

Time-elapsed recruitment video of peptide 1 (Video S1) (AVI).

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