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. 2025 Aug 25;10(35):40056–40065. doi: 10.1021/acsomega.5c04844

Raman Spectroscopy and FRAP Analysis on Density Heterogeneity and Diffusion Behavior of Complex Coacervation by Arginine-Rich Dipeptides and Poly‑A RNA

Ya Tian †, Qiang Zhang ‡, Jiangtao Li †, Motosuke Tsutsumi §,∥, Tomoko Inose ⊥, Farsai Taemaitree †, Kenji Hirai †, Kohsuke Kanekura #,*, Hiroshi Uji-i †,⊥,∇,*
PMCID: PMC12423859  PMID: 40949254

Abstract

Membraneless organelles (MLOs), formed through liquid–liquid phase separation (LLPS), are vital for various cellular functions, including gene expression and RNA metabolism. Destructions of LLPS dynamics, often due to aberrant condensate formation, are implicated in neurodegenerative diseases. Polypeptides composed of repeated dipeptides, namely arginine-rich dipeptide repeat proteins, poly­(proline-arginine) (poly-PR) and poly­(glycine-arginine) (poly-GR), translated from mutated C9ORF72 repeats, undergo LLPS and disrupt RNA and protein homeostasis. Although these peptides have similar net charges, they exhibit different diffusion behaviors in liquid droplets, indicating that sequence-specific charge distribution may influence LLPS dynamics. In this study, we combined fluorescence recovery after photobleaching (FRAP) and Raman spectroscopy to examine the density heterogeneity and diffusion behavior of LLPS droplets formed by arginine-rich dipeptides and homopolymeric adenine RNA (poly-A RNA). We analyzed (PR)20, (P4R4)5, (same components but different charge pattern), and (GR)20 (same net charge as (PR)20, but different sequence). Our results showed that droplets formed with different dipeptides displayed heterogeneous diffusion behaviors, which strongly correlated to internal density gradients within individual droplets detected by Raman spectroscopy. This suggests that charge patterning, not just net charge, critically influences LLPS dynamics. FRAP results of small dye molecules with different net charges align with the concentration gradient of RNA and dipeptides obtained by Raman spectroscopy. Raman spectroscopy combined with FRAP provides a powerful approach for revealing the biophysical properties of biomolecular condensates.

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Introduction

Membraneless organelles (MLOs), or biomolecular condensates formed through liquid–liquid phase separation (LLPS) have attracted significant attention over the past 15 years. These highly dynamic liquid droplets, composed of various charged biological macromolecules, such as proteins and nucleic acids, are involved in numerous essential biological processes, including protein localization, supramolecular assembly, gene expression, gene transcription, RNA metabolism, minimizing cellular noise, genome packaging control, and cell-cycle control. − In contrast, LLPS-driven condensation can also result in the formation of undesirable protein aggregates, disturbing the dynamics and functions of MLOs. − Such dysfunctions are linked to insoluble protein aggregate observed in the neurons of patients with neurodegenerative diseases and to abnormal MLO dynamics caused by toxic dipeptides in amyotrophic lateral sclerosis (ALS). Thus, understanding the fluidity differences of liquid droplets is crucial for elucidating the role of LLPS homeostasis in disease progression. Currently, fluorescence recovery after photobleaching (FRAP) is a widely used method to address LLPS dynamics both in vivo and in vitro.

LLPS is driven by multiple weak interactions, including electrostatic, cation−π, π–π, hydrophobic interactions, and hydrogen bonding. , These interactions determine droplet formation and stability and influence their dynamic properties. The combination of oppositely charged polymers can create an electrostatic (Galvani) potential between the droplets and their surrounding media, a phenomenon supported by liquid-state theory and confirmed via zeta potential measurements. Recently, many studies have focused on liquid–liquid phase separation achieved by mixing RNA with positively charged polypeptides. These electrostatic gradients may significantly influence the behavior and physical properties of phase-separated droplets.

Polypeptides composed of repeated dipeptides, namely, arginine-rich dipeptide repeat proteins, such as poly­(proline-arginine, PR) and poly­(glycine-arginine, GR), produced via repeat-associated non-ATG (RAN) translation of the mutated chromosome 9 open reading frame 72 (C9ORF72) gene, are highly toxic in vitro and in vivo and contribute to ALS and frontotemporal dementia (FTD). − These dipeptides interact with RNA and proteins through abnormal LLPS, disturbing the homeostasis of RNA , and protein metabolism. , Both poly­(PR) and poly­(GR) are positively charged and accumulate in the nucleolus, impairing nucleolar dynamics and inhibiting rRNA processing and protein translation. , Despite similar propensities for LLPS, poly­(GR) exhibits slower diffusion than poly­(PR) both in cellular and in vitro, , though the reason for these differences remains unclear. In vitro, arginine-rich dipeptides undergo complex coacervation with RNA via electrostatic and π-system interactions. Recent studies suggest that variations in charge distribution along the dipeptide sequence significantly affect binding energies and droplet dynamics, even when the net charge is identical. ,

Although prior fluorescence microscopic studies using fluorescently labeled biomolecules have revealed different droplet dynamics in vitro, the underlying causes of these differences remain poorly understood. Given the charged nature of the biopolymers within droplets, their diffusion behavior may vary. Understanding how charge properties influence droplet dynamics is key to uncovering these mechanisms.

To address these questions, we prepared and analyzed LLPS droplets formed by arginine-rich dipeptides with distinct charge properties using Raman spectroscopy and FRAP analysis. Raman spectroscopy, a molecular vibrational technique capable of providing the internal composition of droplets, has proven powerful in elucidating molecular interaction in LLPS system. − In this study, we investigated droplets formed by mixing homopolymeric adenine (poly-A RNA) with three arginine-rich dipeptides: (PR)20 (20 repeats of proline and arginine), (P4R4)5 (five repeats of four proline and four arginine, with the same net charge and components but different charge patterning), and (GR)20 (20 repeats of glycine and arginine, with the same net charge and charge patterning but different components and weaker charge separation). Using FRAP and Raman spectroscopy, we observed spatial density gradients and heterogeneous diffusion dynamics within droplets, consistent with our previous results. We observed higher density of both fluorescently labeled RNA and positively charged polypeptides at the center region compared to that at the near boundary within individual liquid droplets by analyzing the Raman intensity maps. Spatially homogeneous Raman intensity ratio between the dipeptide and RNA within individual liquid droplets indicated the formation of stable RNA-dipeptide complexes. FRAP analysis of fluorescently labeled RNA and various small dye molecules with different net charges further indicated slower diffusion at the center region compared to that at the near boundary within individual liquid droplets. Further analysis in the ratio of center-to-boundary recovery time between (GR)20 and (PR)20 droplets suggests that only negatively charged fluorescein could form stronger interaction with (GR)20 compared to (PR)20 and (P4R4)5. Spatially heterogeneous diffusion dynamics of molecules within droplets observed with FRAP analysis strongly correlate with droplet density measured by Raman spectroscopy. These findings demonstrate that the combination of FRAP and Raman spectroscopy offers complementary insights into how the charge properties of droplet components influence the complex dynamic behaviors of LLPS-based biomolecular condensates.

Experimental Section

Chemicals

The peptides (PR)20, (GR)20, and (P4R4)5 were chemically synthesized by GenScript (Piscataway, NJ) with a purity greater than 85%. Poly-A RNA (700–3500 kDa) was purchased from Sigma-Aldrich and used without further purification. TAMRA-rA15 (5-carboxy-tetramethyl-rhodamine (TAMRA)-labeled poly-A15 (15 repeats of adenine) was synthesized and purified by Fasmac (Kanagawa, Japan).

Phase Separation Assay

Stock solutions of arginine-rich dipeptides [(PR)20, (GR)20 or (P4R4)5] were prepared at ∼1 mM in phosphate-buffered saline (PBS; 049-29793, Wako, pH 7.3, [Na+] = 152 mM). A poly-A RNA stock solution was prepared at 0.55 mg/mL in the same PBS. To estimate the dipeptide concentration of each solution, the absorbance at 205 nm (A205) of the 12-fold diluted stock solution was measured using a UV–vis spectrophotometer (VL0000D0, Thermo Scientific), based on the absorbance contribution of peptide bonds and side chains. , Stock solutions of small dye molecules, TAMRA, Rhodamine B, and fluorescein, were also prepared at 1 mM in PBS.

For Raman spectroscopy, fluorescently nonlabeled droplets were prepared. Shortly, 45.5 μL of the poly-A RNA stock solution was diluted with 136.5 μL of PBS buffer. Then, 18 μL of the peptide stock solution was added, and the mixture was vortexed. The mixture was transferred to an 8 well noncoated coverslip (Matsunami) and incubated for 3 h at room temperature.

For the FRAP measurement of fluorescently labeled RNA, 1 μL of TAMRA-rA15 (0.55 mg/mL) was mixed with 44.5 μL of the poly-A stock solution in 136.5 μL of PBS, followed by vortexing with 18 μL of the peptide stock solution. The mixture was incubated in an 8 well noncoated coverslip for 3 h at room temperature to form fluorescently labeled droplets. For FRAP analysis of small dyes, 1 μL of the stock solution of TAMRA, Rhodamine B, or fluorescein was mixed with 45.5 μL of the poly-A stock solution in 135.5 μL of PBS, then vortexed with 18 μL of the (PR)20 or (GR)20 stock solution and incubated at room temperature for 3 h to form droplets labeled with free dye molecules. The final concentrations of poly-A RNA were approximately 0.125 mg/mL in all solutions.

FRAP Analysis

FRAP experiments were conducted using a confocal microscope (A1Rsi, Nikon) equipped with a 60× water-immersion objective (PlanApo, NA 1.2, Nikon), following the protocols described previously. , Fluorescein was excited by a 488 nm laser, and the emission signal was detected through a 525/50 bandpass filter. TAMRA, RhB, and TAMRA-rA15 were excited by a 561 nm laser, and the emission signal was detected through a 593/46 bandpass filter. A small area (approximately a 1.0 μm diameter circle) was selected within a liquid droplet. The central or inner boundary region of individual droplets was bleached with 8 scans using 10% (approximately 2 μW, 488 nm) laser power for fluorescein fluorescently labeled droplets, or 19% (approximately 2 μW, 561 nm) laser power for TAMRA, RhB, and TAMRA-rA15 fluorescently labeled droplets, and the fluorescence recovery was recorded over time. Snapshots were then collected using as low laser power as 0.1% (at 10 nW range) every 1 s for 34 s (for the small dye molecules fluorescently labeled droplets showing fast fluorescence recovery) or every 5.0 s for 2.5 min (for TAMRA-rA15 fluorescently labeled droplets showing slow fluorescence recovery). The relative fluorescence intensities in the bleached area were normalized using the average intensity of three images measured before photobleaching. The normalized intensities were analyzed using a fitting equation for a single exponential association model and the fluorescent half-recovery time (τ1/2) was calculated following previously reported methods. − Statistical analyses were performed by a Student’s t-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Raman Spectroscopy

Raman measurements were performed using a home-built Raman microscope based on an inverted microscope (TiU, Nikon) with a 60× air objective (PlanApo, NA 0.95, Nikon), a spectrograph (iHR330, HORIBA) and a CCD camera (Newton DU920P-BEX2-DD, Andor Technology). A 638 nm laser (638-06-MILD-180, Cobolt) was used as the excitation source. Raman scattering was collected through a confocal pinhole and a long-pass filter (BLP01-647R, Semrock). Single-point Raman spectra were recorded at the center region of individual droplets with 1 s exposure time and 300 accumulations. Raman intensity mapping of individual droplets was performed using Omega software (AIST-NT) with a 1 s exposure time and 50 accumulations per pixel. Reference Raman spectra of dipeptides and poly-A RNA in Milli-Q water (18 MΩ cm, Milli-Q System, Millipore) were acquired under the same conditions with 1 s exposure and 1000 accumulations. Raman spectra in each mapping were normalized using spectra obtained from the PBS surrounding the droplets. A wavelet transform was applied to each spectrum to optimize denoising and background removal (see Figure S1).

Results and Discussion

Raman Spectroscopic Analysis of Liquid Droplets

A mixture of dipeptides and poly-A formed spherical droplets ranging from a few to 10 μm in diameter after 3 h of incubation. Although some nonspherical droplets were present, the spherical droplets maintained their shape for several hours on the glass surface at room temperature and were used for Raman spectroscopy in this study. To elucidate the vibrational modes and potential interactions between the dipeptides and poly-A within the droplets, Raman spectroscopy was performed on droplets of approximately 6 μm in diameter. Figure a,b shows representative Raman spectra of droplets composed of (PR)20/poly-A and (GR)20/poly-A, respectively. For comparison, the reference Raman spectra of (PR)20, (GR)20, and poly-A are also presented in Figure . The Raman spectra of the liquid droplets display Raman peaks associated with both arginine-rich dipeptides and poly-A RNA (peak assignments are summarized in Table ). − A prominent feature of the spectra is a peak around ∼1580 cm–1, which is attributed to the υ (CC) stretching mode of adenine of poly-A. Additionally, a peak at 1450 cm–1 was observed in (PR)20-containing droplets and a similar peak around 1445 cm–1 appeared in droplets with (GR)20. These peaks likely correspond to CH2 /CH3 deformation modes from the side chain of the arginine-rich dipeptides. ,, Notably, the peak at 1437 cm–1, associated with the N–H deformation of the guanidinium moiety in arginine, was suppressed upon droplet formation. This suppression suggests hydrogen bonding between arginine residues and the adenine bases of RNA.

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Single-point Raman spectroscopy of the liquid droplets. Comparison of typical Raman peaks observed in liquid droplets with reference Raman spectra of dipeptides and poly-A RNA. (a) Reference Raman spectra of poly-A RNA (red line) and (PR)20 (blue line) (top), and the Raman spectrum measured at the center of a (PR)20/poly-A droplet (bottom). (b) Reference Raman spectra of poly-A RNA (red line) and (GR)20 (blue line) (top), and the Raman spectrum measured at the center of a (GR)20/poly-A droplet (bottom). Dashed lines indicate characteristic peaks corresponding to the dipeptide (green and black dashed lines) and poly-A RNA (red dashed lines), respectively. The peak at 1437 cm–1, attributed to N–H deformation of the guanidinium moiety of arginine, is observed in both (PR)20 and (GR)20.

1. Assignment of the Characteristic Experimental Vibrations of Each (PR)20/Poly-A RNA Droplet to Specific Vibrational Modes.

Peak (cm–1) Assignment
1580 Adenine, CC (V base), in plane
1510 Adenine, vibrations of benzene, skeletal stretching
1450 Arginine, CH2/CH3 deformation modes
1437 Arginine, N–H deformations of the guanidinium moiety of arginine
937 Arginine, C–C; N–C–N; C–N
843 Proline, skeletal vibrations
795 Adenine, CC
726 Adenine, ring-breathing mode vibration
597 Bending of COO–
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Since spontaneous Raman scattering intensity is proportional to the molecular concentration, the density of the liquid droplet can be analyzed using Raman spectroscopy. From the UV–vis spectroscopy, the concentrations of (PR)20 and (GR)20 in the stock solutions were estimated to be 1.20 and 1.06 mM, respectively (Figure S4). By comparing the Raman spectra of droplets formed from these dipeptides with an equal amount of poly-A, we found that the Raman scattering intensity from (GR)20/poly-A droplets was 1.1 to 1.2 times higher than that from (PR)20/poly-A droplets (Figure S5), indicating a higher density of (GR)20/poly-A complexes within the droplets.

To gain further insight into the spatial composition of the droplets (Figure a), Raman mapping was performed across individual droplets (Figure b). Figure c,d shows Raman intensity maps along the x- and z-directions, respectively, using the peak intensity at 1580 cm–1 (assigned to the υ (CC) of poly-A) to visualize the RNA distribution. No Raman peaks were detected outside the droplets, while characteristic peaks of (PR)20 and poly-A were clearly observed within. The Raman maps revealed slightly compressed spherical droplets, measuring approximately 5 μm in diameter in the xy-plane and ∼3.5 μm in the z-direction for the droplet shown in Figure c,d. Note that, in Figure c, the spectrum at position #2 corresponds to the edge of the droplet, where the excitation laser focus partially overlaps the inside and outside of the droplet. In contrast, at position #3, the laser is fully focused inside. The Raman intensity of the υ (CC) peak at the center region of the droplet (position #7) was significantly higher than that near the inner boundary (positions #3–4), indicating a higher density of poly-A at the droplet center. Similar intensity profiles were observed at 1450 or 1445 cm–1, corresponding to CH2/CH3 deformation modes of the dipeptides (Figure S2).

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Raman mapping of liquid droplets composed of (PR)20 and poly-A RNA. (a) A typical optical transmission image of (PR)20 /poly-A RNA droplets. Only spherical droplets were analyzed in this study. (b) Schematic illustration defining the axis used for Raman microscopy measurements. (c) Raman intensity map of a liquid droplet by scanning along the x-axis (top). Raman scattering spectra collected at various positions: one spectrum from outside of the droplet (position #1) and a series of spectra from the edge to the center of the droplet (positions #2 to #7) (bottom). The light green background at 1580 cm–1, which is attributed to υ (CC), indicates the presence of RNA. (d) Raman intensity map of the droplet obtained by scanning along the z-axis. (e–g) Intensity ratio of the Raman peaks at 1450 or 1445 cm–1 (dipeptide related) and 1580 cm–1 (RNA related), I 1450/I 1580, or I 1445/I 1580 acquired along the x-axis (top) within droplets composed of (PR)20/poly-A RNA (e), (P4R4)5/poly-A RNA (f), and (GR)20/poly-A RNA droplets (g), respectively. Normalized Raman spectra at positions #3 and #7 are shown for comparison (bottom).

Despite the presence of concentration gradients for RNA and dipeptides, the intensity ratio map between the peaks at 1450 cm–1 (dipeptide related) and 1580 cm–1 (poly-A related), I 1450/I 1580, remained relatively homogeneous across the (PR)20 droplets (Figure e, top). (See Figure f,g for the corresponding intensity ratio maps across the (P4R4)5 and (GR)20 droplets, respectively.) This uniform ratio suggests the formation of stable RNA-dipeptide complexes, likely driven by strong interaction between them, electrostatic, cation−π, π–π, and/or hydrophobic interactions. −

We examined the density heterogeneity of (P4R4)5, which has the same net charge as (PR)20 but different charge patterning, and in (GR)20 which shares both the same net charge and charge patterning as (PR)20 but has insufficient charge separation and different amino acid components. Raman intensity maps at 1580 cm–1 (RNA related) and 1450 cm–1 (dipeptide related) for (PR)20/poly-A (Figure S2a), (P4R4)5/poly-A (Figure S2b), and 1580 and 1445 cm–1 for (GR)20/poly-A droplets (Figure S2c) reveal clear concentration gradients of poly-A RNA and dipeptides from the center to the inner boundary of the droplets. These suggest that arginine-rich dipeptides play a crucial role in forming a hierarchical structure within liquid droplets. Interestingly, although (GR)20, (PR)20, and (P4R4)5 share the same isoelectric point (e.g., pI = 12.37), the peak intensity at 1580 cm–1 is significantly higher in (GR)20 droplet compared to (PR)20 and (P4R4)5 droplet (Figure S3), indicating a greater accumulation of RNA in (GR)20 droplet. This is particularly notable given that the stock concentration of (GR)20 (1.06 mM) is lower than that of (PR)20 (1.20 mM) (Figure S4).

Despite both peptides containing arginine residues and having the same pI, the difference in charge distribution and structural flexibility likely influences their binding behavior to RNA. In (PR)20, the cyclic structure of proline spatially separates arginine residues, weakening electrostatic interactions with the negatively charged RNA backbone and resulting in partial binding with RNA. In contrast, (GR)20 contains glycine, of which a minimal side chain (a single hydrogen atom) does not disrupt arginine clustering. This results in a block-like positive charge pattern that strengthens electrostatic attraction to poly-A RNA. ,, Furthermore, the guanidinium groups of arginine in (GR)20 can participate in a variety of interactions, including electrostatic interactions to phosphate backbone and cation−π and π–π stacking interactions with adenine bases. These diverse interaction modes allow individual arginine residues to engage with multiple RNA sites, supporting cooperative rather than competitive binding. , Additionally, the structural flexibility of (GR)20 due to glycine spacers enhances its ability to conform to RNA and form π–π interactions more than the rigid proline-containing (PR)20. ,, This structural flexibility likely contributes to the higher RNA recruitment in (GR)20-containing droplets, as reflected by the increased Raman scattering intensity associated with RNA (Figure S5a). Therefore, despite its lower initial concentration, (GR)20 exhibits a stronger capability to accumulate RNA within droplets. This is likely driven by its clustered positive charges, conformational flexibility, and ability to form multivalent interactions.

Diffusion Behavior of Poly-A RNA in Liquid Droplets

To elucidate the molecular diffusion dynamics within liquid droplets, we performed FRAP measurements. In the initial set of experiments, FRAP was conducted using fluorescently labeled poly-A RNA (TAMRA-rA15) to monitor the diffusion behavior of arginine-rich dipeptide/RNA complexes (Figure ). For this purpose, TAMRA-rA15 was mixed with unlabeled poly-A at a concentration of 2.25 wt % and incubated with the dipeptides. Figure a presents representative fluorescence snapshots of (PR)20/poly-A droplets during FRAP measurements, showing fluorescence recovery after photobleaching at the central region (Figure a top) and near the inner boundary (Figure a bottom) of the droplet. The corresponding fluorescence recovery curves as a function of time are shown in Figure b. The average half-recovery time (τ1/2) was estimated to be 37.6 ± 4.4 s at the center and 32.1 ± 3.3 s near the inner boundary (Figure b,e, respectively). Similar measurements were conducted for (P4R4)5/poly-A (Figure c) and (GR)20/poly-A droplets (Figure d), where comparable spatial trends were observed (Figure e). In all cases, fluorescence recovery was consistently slower at the central region than at the inner boundary, indicating spatially heterogeneous diffusion dynamics of the dipeptide/RNA complexes. This slower recovery correlates with the higher concentration of the complexes at the droplet center, as also revealed by Raman spectroscopy (Figures and S2). In all liquid droplets labeled with TAMRA-rA15, the fluorescent recovery of TAMRA-rA15 molecules was found not to fully recover, as shown by the recovery curves for all droplet types (Figure b–d). This partial recovery suggests that a fraction of dipeptide/RNA complexes diffuse slowly within the liquid droplets during the limited observation time and/or the intensity did not recover due to the photobleaching effect of the low amount of TAMRA-rA15. Furthermore, the intermediate fluorescence recovery of TAMRA-rA15 at the center region is slightly higher than that at the near boundary within liquid droplets (Figure b–d). These results correspond to the local density gradients across the droplet regions.

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Diffusion of poly-A RNA in liquid droplets. (a) Fluorescence snapshots of (PR)20/poly-A RNA droplets before and after photobleaching. (b–d) Averaged FRAP curves at the center (blue line) and the inner boundary (red line) of droplets composed of (PR)20/poly-A RNA (b), (P4R4)5/poly-A RNA (c), and (GR)20/poly-A RNA droplets (d), respectively. (e) Averaged half-recovery time (τ1/2) at the center (blue bars) and inner boundary (orange bars) of each dipeptide. (f) Ratio of the recovery time at the center to that at the boundary for TAMRA-rA15 in (PR)20 /poly-A RNA (black), (P4R4)5/poly-A RNA (purple), and (GR)20 /poly-A RNA (pink) droplets. Asterisks indicate statistical significance based on t-test: *, p < 0.05, **, p < 0.01, ***, p < 0.001, NS: not significant (p > 0.05).

The τ1/2 values for TAMRA-rA15 in (P4R4)5/poly-A droplets were estimated to be 42.9 ± 9.5 s at the center and 36.1 ± 5.6 s at the boundary (Figure c,e), similar to the values observed with the (PR)20 dipeptide. This suggests that the diffusion behavior of the dipeptide/RNA complexes is not strongly affected by the charge distribution pattern of the dipeptide repeat proteins. In contrast, τ1/2 values in (GR)20/poly-A droplets were significantly longer; 56.9 ± 19.8 s at the center and 47.4 ± 11.6 s at the boundary (Figure d,e). This increase is likely due to the higher density of (GR)20/poly-A droplets, as confirmed by Raman spectroscopy (Figure S5a). Furthermore, the ratios of average recovery half-times between the center and boundary remained consistent across all droplet types (Figure f). These results indicate that the diffusion behavior of poly-A RNA in arginine-rich dipeptide/poly-A RNA droplets is primarily governed by the droplet density rather than the charge pattern of the dipeptides.

Diffusion Behavior of Small Molecules in Liquid Droplets

In the second set of FRAP measurements, we investigated the diffusion behavior of small molecules within droplets using three types of fluorescent dyes: TAMRA (Figure a), Rhodamine B (RhB) (Figure b), and fluorescein (Figure c). These dyes were selected to mimic the diffusion behavior of small biomolecules or drug-like compounds. In PBS (pH 7.4), TAMRA has a neutral net charge, RhB carries a positive charge, and fluorescein is negatively charged. , Prior to droplet formation, the dyes were dissolved in PBS and mixed with a precursor solution containing nonlabeled poly-A and the dipeptides, yielding a final dye concentration of 5 μM. Figure d–f shows representative FRAP snapshots of the dyes at the center and boundary of a (PR)20/poly-A droplet. Notably, the fluorescence intensity of the negatively charged dye (fluorescein) recovered almost completely within 11 s after photobleaching (Figure f), whereas the bleached regions for TAMRA and RhB remained visible (Figure d,e). This observation indicates that the negatively charged dyes diffuse more rapidly than the neutral or positively charged ones. In all cases, all small dye molecules showed almost full fluorescent recovery (Figures g–i, S6d–f, and S7d–f). This suggests that small dye molecules diffuse quickly within the liquid droplets in a very short observation time. When comparing the recovery curves of the dipeptide/RNA complexes and small dye molecules in (PR)20 droplets (Figures b and g–i), the large molecular size of dipeptide/RNA complexes showed slow recovery, while the small dye molecules did quick recovery in the liquid phase. This suggests that the large molecular size of dipeptide/RNA complexes leads to molecular crowding due to the volume exclusion effect.

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Diffusion of small molecules in liquid droplets. Chemical structures of TAMRA (a), Rhodamine B (b), and fluorescein (c), respectively. Fluorescence snapshot after photobleaching in the center region and inner boundary of (PR)20/poly-A RNA droplets containing TAMRA (d), Rhodamine B (e), or fluorescein (f). Averaged FRAP curves at the center (blue) and inner boundary (red) of (PR)20/poly-A RNA droplets containing TAMRA (g), Rhodamine B (h), and fluorescein (i), respectively.

The corresponding fluorescence recovery curves for the three dyes are shown in Figure g–i, respectively. Blue lines represent the FRAP curves at the droplet center, and red lines correspond to the inner boundary of the (PR)20/poly-A droplet. (See Figures S6 and S7 for the corresponding snapshots and recovery curves in (GR)20 and (P4R4)5 droplets, respectively.) The averaged τ1/2 values for each dye in droplets containing (PR)20, (P4R4)5, or (GR)20 are summarized in Figure a–c. Consistent with the results obtained using fluorescently labeled poly-A RNA (Figure ), fluorescence recovery was generally faster at the droplet boundary than at the center, regardless of the charges of the dye or dipeptide sequence. This suggests that the diffusion of small molecules is generally enhanced near the droplet boundary. For (PR)20-containing droplets, the averaged τ1/2 of TAMRA at the center was estimated to be 2.5 s. For fluorescein, τ1/2 was slightly shorter at 1.5 s, while RhB exhibited a significantly longer τ1/2 of 5.6 s (Figure a). This charge-dependent trend in recovery time was consistent across both droplet regions and different dipeptide sequences. Notably, the recovery times in (GR)20 droplets were approximately twice as long as those in (PR)20-containing droplets (Figure a,c), likely due to the higher density of (GR)20-containing droplets, as discussed in Figure .

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Averaged recovery time of small molecules in liquid droplets. (a–c) Averaged half-recovery time at the center (blue) and inner boundary (orange) of droplets containing (PR)20 (a), (P4R4)5 (b), and (GR)20 (c). (d) Ratio of the averaged recovery half-time between the center and boundary in (PR)20 /poly-A RNA (black), (P4R4)5/poly-A RNA (purple), and (GR)20 /poly-A RNA (pink) droplets. Asterisks indicate statistical significance based on t-test: *, p < 0.05, **, p < 0.01, ***, p < 0.001, NS: not significant (p > 0.05).

Finally, the ratio of recovery time between the droplet center and boundary for TAMRA and RhB showed no significant difference across (PR)20, (P4R4)5, and (GR)20-containing droplets. However, for fluorescein, this ratio varies significantly between the different dipeptide droplets (Figure d), suggesting a stronger binding between negatively charged molecules and (GR)20 compared to (PR)20. Previous studies using zeta potential measurements and simulations have shown that such droplets are generally negatively charged. ,, This could explain the observed charge-dependent diffusion behavior of the fluorescent dyes. These differences in diffusion behavior are likely due to electrostatic interactions within the droplets, where negatively charged fluorescein is repelled and thus diffuses more rapidly, while positively charged RhB experiences attractive interactions and, therefore, diffuses more slowly. Thus, by tuning the charge of drug molecules to align with the electrostatic environment of the droplets during drug design, it may be possible to retain their enrichment within the droplets while reducing diffusion hindrance. In combination with droplet-disrupting amines, such as 1,6-hexanediol (1,6-HD), this strategy could enhance drug bioavailability and open up novel therapeutic avenues.

Collectively, our Raman and FRAP measurements reveal heterogeneous dynamics and hierarchical structure within liquid droplets, consistent with an internal concentration gradient of RNA. The complex diffusion behavior observed is primarily driven by the formation of stable RNA–dipeptide complexes. Importantly, both the overall charge and density variations within droplets, arising from interactions with arginine-rich dipeptides, should be taken into account when analyzing diffusion processes in these systems.

Conclusions

In this study, we investigated the molecular organization and diffusion behavior within arginine-rich dipeptide/poly-A RNA condensates using Raman spectroscopy and fluorescence recovery after photobleaching (FRAP). Raman spectroscopic analysis revealed that the dipeptides (PR)20 and (GR)20, when mixed with poly-A RNA, form spherical liquid droplets enriched with RNA–dipeptide complexes. Raman intensity mapping demonstrated a concentration gradient of poly-A RNA, with higher density at the droplet center. This suggests the formation of a hierarchical internal structure within droplets, likely governed by intermolecular interactions such as hydrogen bonding, cation−π interactions, and electrostatic forces. FRAP measurements further revealed spatially heterogeneous diffusion dynamics inside the droplets. Labeled poly-A RNA exhibited slower fluorescence recovery at the center compared to the periphery, in agreement with the Raman-based density distribution. Interestingly, the diffusion behavior was more strongly influenced by droplet density than by the specific charge pattern of the arginine-rich dipeptides. Droplets formed with (GR)20 showed significantly slower recovery times than those with (PR)20, consistent with their higher molecular density. Additionally, the diffusion of small fluorescent dyes varied with their net charge. Negatively charged fluorescein diffused more rapidly than positively or neutrally charged dyes, highlighting the charge-dependent permeability of the droplet environment. This permeability varied across dipeptide sequences and droplet regions, suggesting selective molecular partitioning based on physicochemical properties. Together, our results reveal that arginine-rich dipeptide/poly-A RNA droplets are not uniform liquid phases but possess a complex internal structure and diffusion behavior shaped by molecular density, charge interactions, and component composition. These findings underscore the importance of considering internal density heterogeneity and charge-driven interactions when studying biomolecular condensates, with implications for understanding the regulation of biochemical processes in membraneless organelles, designing synthetic condensates for biomedical applications, and developing potential therapeutic strategies for treating neuronal diseases.

Supplementary Material

ao5c04844_si_001.pdf (605.2KB, pdf)

Acknowledgments

This work was financially supported by JSPS Kakenhi (Grants 25H00827, 24K02187, 24K21710, 24K21713, 23H04877, 23K26496, 23K17856, 22H00328, 21H04634), JSPS J-PEAKS awarded to Hokkaido University, the Internal Fund of KU Leuven (C14/23/090), and the Research Foundation Flanders (FWO G022724). Platforms for Advanced Technologies and Research Resources “Advanced Bioimaging Support” are acknowledged. Y.T. acknowledges the Hokkaido University President’s Fellowship.

Glossary

Abbreviations

(PR)20

20 repeats of proline-arginine

(GR)20

20 repeats of glycine-arginine

(P4R4)5

variants of (PR)20, five repeats of proline-arginine

MLO

membrane-less organelle

ALS

amyotrophic lateral sclerosis

FTD

frontotemporal dementia.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04844.

  • Normalization of Raman spectra in the droplets. Raman scattering intensity maps of the liquid droplets sweeping in the x-direction. Peak intensity at 1580 cm–1 in the droplets. The UV absorbance spectra of dipeptide solutions. The Raman intensity of liquid droplets, Diffusion of small molecules in (GR)20/poly-A droplets. Diffusion of small molecules in (P4R4)5/poly-A droplets (PDF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

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