Optically driven control of mechanochemistry and fusion dynamics of biomolecular condensates via thymine dimerization

Domain: physics
Published: 2026-03-26
Editorial outcome: major_revision

Abstract
Phase-separated biomolecular condensates are functional elements in cells, contribute to protocell formation in prebiotic systems, and represent a distinct class of soft matter. Controlling condensate mechanochemistry is critical for function and material properties. Although photochemical processes are widespread in nature and can be harnessed in engineering, it remains unclear how condensate formation affects photochemistry, and conversely how photochemistry alters condensate dynamics. Using scanning probe microscopy combined with UV-controlled photochemistry and optical imaging, we develop assays to probe mechanical transitions and fusion dynamics in condensate droplets, revealing that UV-induced thymine dimerization alters condensate nucleation and coalescence. Depending on the frequency and topological arrangement of thymine dimers, particularly the balance between inter- and intrachain crosslinks, UV can drive transitions from liquid-like to solid-like states or induce aggregates. UV also promotes arrested fusion and stable compartmentalization of droplets, resilient to environmental changes and with implications for prebiotic chemistry and bio-inspired engineering.

Introduction
Biomolecular condensates formed through liquid-liquid phase separation (LLPS) play key roles in various cellular processes, ranging from gene regulation to stress granule formation 1–5. These condensates assemble through multivalent interactions among macromolecules such as nucleic acids and proteins6–9, and while physiological condensates are often liquid-like, they can exhibit diverse material and mechanical properties, particularly upon aging or under stress conditions that alter their interactions10–15. The strength, density, and topological arrangement of interactions, along with the intrinsic mechanical properties of the polymer chains, are critical determinants of condensation and the resulting mechanical phenotype16. Such variations in condensate material state may directly influence molecular transport and chemical reactions within their microenvironments, effects that are central to cellular biochemistry and exploitable for reaction control in engineered systems6,17–21. Modulation of the mechanical properties of condensates is typically achieved by introducing new molecules (e.g., crosslinkers) or adjusting molecular concentrations, approaches that inherently alter condensate composition, making it difficult to disentangle the effects of mechanics and chemistry22.

Light can be exploited to modulate material properties in soft and polymeric systems23,24. Yet, light‑triggered control over the mechanical state of condensates, achieved without significantly altering their chemical composition, offers a largely unexplored route for engineering condensates. One particularly relevant form of light stimulus is ultraviolet (UV) radiation, which is both a major cause of DNA damage through the formation of covalently bonded pyrimidine dimers such as thymine dimers (TT) and a critical factor in the emergence of life on Earth25,26. TT dimers disrupt normal DNA replication27 and transcription28, leading to mutations that can initiate various diseases, and are the primary cause of skin cancer29. Cells recognize and attempt to repair these lesions30; however, persistent or improperly repaired TT dimers can give rise to short stretches of single-stranded DNA (ssDNA), ultimately contributing to genomic instability and carcinogenesis31.Furthermore, UV radiation was abundant on early Earth and contributed to the synthesis of building blocks of life32, while coacervate condensates of these building blocks are also considered plausible protocellular structures33. The droplets feature a highly dynamic molecular environment34, and are permeable to molecules from their surroundings with certain degrees of selectivity, enabling chemical synthesis and molecular recognition (e.g., receptor-ligand interactions) required for life35. Nucleic acids, known for their propensity to form condensates, were likely key components of protocells33. Because coacervates lack lipid membranes, these protocellular structures provide only limited shielding of their contents from solar UV. It remains unclear, however, what UV does to (nucleic acid) coacervates and whether it can shape their structural mechanics.

A technical challenge in studying the mechanochemistry of condensate droplets is the difficulty in probing viscoelastic and fusion properties as they undergo liquid-to-solid transitions. Active microrheological characterization of condensate droplets is mostly performed utilizing optical tweezers (OT)36,37, which are constrained to low-force regimes and generally rely on trapped beads. Micropipette aspiration is also constrained by achievable force (suction pressure) and experimental throughput38,39. Recently, scanning probe microscopy (SPM), a technique widely used in mechanobiology and materials science40, has been applied to characterize the material properties of condensates22,41,42, covering a significantly higher range of forces and eliminating the need for exogenous particles in the system. Yet, SPM has never been applied to study droplet-droplet fusion, a gap that, if addressed, could enable direct insights into condensate mechanics and interactions.

Here, we introduce a photochemical strategy to precisely modulate condensate mechanics using UV-induced thymine dimerization, enabling control over mechanical properties while preserving chemical composition that is, without adding new molecules and without changing atomic composition. Using poly-thymine single-stranded DNA (dT40, Table S1) and poly-L-lysine (PLL) as a model system, we demonstrate how this strategy drives transitions among distinct mechanical states, from liquid-like droplets to more rigid, gel- or solid-like networks. To capture these mechanical changes, we developed SPM-based assays combined with optical imaging for label-free characterization of condensate mechanics and fusion dynamics. These transitions lead to dramatic changes in fusion behaviour, ultimately driving compartmentalization, a process relevant to prebiotic chemistry and bio-inspired materials design. We attribute these observations to the formation and topological arrangement of UV-induced TT dimers, which can be local, long-range, or occur between chains. Our study provides a compelling example in which a photochemical reaction alters the condensed phase, and conversely, condensation modulates photochemical reaction outcomes.

Results
UV irradiation affects the phase separation behaviour and promotes arrested fusion droplets
We first explored the phase diagram of PLL/dT40 by varying component concentrations and selected a 1:1 PLL:dT40 ratio at 30 µM of each component for subsequent analyses (Figures 1A and S1), as this condition produced micron-sized droplets that were well suited for mechanical measurements and imaging. Prior to investigating the effects of UV irradiation, we characterized the baseline phase separation behaviour of PLL/dT40 under varying salt conditions, from 0 M to 1 M. No droplets formed at either extreme: high salt screens electrostatic interactions below the threshold for phase separation, while low salt favors tight 1:1 heterodimers with a strong positive net charge (due to charge asymmetry -40 vs. +150), preventing further association. Intermediate salt concentrations, by contrast, allow molecules to exchange partners within a liquid environment47,48, promoting spherical condensates of varying size. The largest droplets were consistently observed at 150 mM KCl (Figure 1B and C), a condition used for the remainder of our study.

For UV treatment, samples were prepared over a total period of 4 hours and placed under a UVC lamp (UVP UVG-11, =254 nm; 1120µW/cm² at the sample) (Figure 1D), with UV illumination applied during the final 30 (+UV30) or 45 (+UV45) minutes49. Spectroscopic analysis indicates that UV induces TT dimerization in dT40 under this exposure condition (Figure 1G, and H). Although the UV illumination times used in this study are much longer than the picosecond timescale required for the formation of intramolecular TT dimers50, it is comparable to the timescale characterizing intermolecular crosslinking49. Phase-contrast microscopy immediately after irradiation revealed altered droplet morphologies with an apparent reduction in circularity (Figure 1E, and F). Similar UV-induced morphological changes were observed with droplets formed on nonsticky surfaces (Figure S2A and S2B), suggesting that observed patterns are primarily driven by UV-induced chemical changes rather than surface adhesion effects (Figure S2C). To confirm that UV irradiation indeed trigger TT dimer formation inside droplets, we performed immunostaining using anti-TT dimer antibody, which verified TT dimerization in dT40 under these exposure conditions (Figure 1I).

Our quantitative analysis revealed that extended UV exposure (from 30 to 45 min) led to a higher occurrence of doublets and clustered configurations (Figure 2A, and B), as well as a significant increase in droplet-droplet contact area, measured by the chord length (L) (Figure 2A, and C). This reflects slower relaxation due to the expected increase in viscoelasticity and allows doublets with larger chord lengths to be more readily captured during imaging. Additionally, our particle analysis revealed a significantly lower circularity index of +UV45 droplets compared to +UV30 and control (Figure 2D). Size distribution analysis of singlet droplets revealed that the control samples exhibited an exponential distribution (and not log-normal51 or power law52 behaviours), consistent with a quench-then-coalesce mechanism53,54. However, +UV30 samples, and more prominently +UV45 samples, exhibited a clear shift to a power-law distribution, suggesting a transition to an addition of mass growth model52 (Figures 2E and S3).

Rheological analysis reveals liquid-to-solid-like transitions upon UV exposure
UV exposure can induce inter- and intra-chain thymine dimers, as confirmed by our spectroscopic and imaging analyses, potentially altering condensate mechanics55,56. To investigate this, we performed oscillatory rheology measurements of the storage modulus (G′) and loss modulus (G″) on control and UV-illuminated samples using a recently developed SPM-based approach22 (Figure 3A, Note S1). Across the entire frequency range examined (1 Hz to 100 Hz), G″ consistently exceeded G′, indicating that the control droplets exhibited predominantly viscous (liquid-like) characteristics (Figure 3B). We note that the mechanical properties of this condensate system are quite robust within the timescale of our study and do not change over tens of minutes.

Following UV treatment, significant changes in the viscoelastic responses were observed (Figure 3C). In +UV30 samples, G′ and G″ increased substantially compared to control and intersected around 20 Hz, indicating a liquid-to-solid-like crossover. At frequencies below 20 Hz, G′ rose from 5 Pa to ~300 Pa, and G″ from 20 Pa to ~600 Pa. Above 20 Hz, G′ rose more steeply, reaching ~4500 Pa at 100 Hz, while G″ reached ~2600 Pa, reflecting dominant elasticity at high frequencies. In +UV45 samples (Figure 3D), a similar trend is seen, but the crossover is shifted to higher frequencies (f > 40 Hz), where differences between G′ and G″ become nonsignificant, suggesting the formation of heterogeneous stiff domains.

These results support a model in which UV exposure drives condensates from a low-stiffness, liquid-like state to a stiff, solid-like state with slow relaxation at moderate exposure (+UV30), and further UV produces a heterogeneous solid-like system where local stiffening increases elasticity but allows faster relaxation in less-crosslinked regions (+UV45). To better highlight differences between conditions, we also plotted the complex modulus (G*) at 10 Hz (Figure 3E), which reveals a systematic increase in viscoelastic stiffness with increasing irradiation duration. Finally, we measured the terminal viscosity of these condensates at T = 25°C by a linear fit of G″ over frequency. Control samples had the lowest viscosity (6.2 ± 0.09 Pa.s), followed by +UV30 (12.08 ± 0.11 Pa.s), while +UV45 condensates showed the highest (140.77± 11.7 Pa.s).

UV irradiation significantly alters nucleation and coalescence processes
UV irradiation induces thymine dimerization in DNA molecules, forming intrachain covalent bonds and, with lower probability, interchain crosslinks49. This competition between intra- and inter-chain interactions can significantly influence condensate formation and growth. Conversely, the prevalence of each interaction type may depend on the condensation stage, as condensation itself can modulate photochemical outcomes. To investigate this phenomenon, we conducted UV irradiation for 45 minutes under two conditions: the "Premix" condition, where UV illumination was applied to dT40 after the addition of buffer and before the addition of PLL, and the "0 hours" condition, where UV exposure was performed immediately after mixing dT40 and PLL. Interestingly, under the "0 hours" condition, no condensates were detected by our optical microscopic analysis after 4 hours. Instead, a large network of interacting components was formed (Figure 4A, middle panel). Real-time phase-contrast imaging, however, revealed the transient formation of small condensate-like droplets, which subsequently transitioned into aggregate-like structures during the UV illumination period (Figure S4A). These structures contained DNA and PLL, as shown by fluorescence analysis (Figure S4B). Micro-Flow Imaging (MFI) analysis revealed the real-time formation of small particles with a size distribution distinct from that of pure condensate systems, suggesting the formation of microaggregates with non-circular geometries (Figure S4).

Interestingly, in the premix condition, we observed condensate formation (Figure 4A, left), with smaller sizes (Figure 4B, and C) and circularity values comparable to control droplets (Figure 4D), indicating no elongation or deformation. Furthermore, premix UV irradiation had minimal impact on condensate mechanics, compared to the control (Figure 4E), which may be attributed to the low occurrence of inter-chain interactions. Consistent with this interpretation, molecular exchange analysis showed strongly reduced DNA exchange in +UV45, and efficient exchange in +UV45_Premix and control (PT40, droplets received no UV illumination) droplets. To demonstrate this, we added 1% TYE-665 tagged dT40 to the dilute phase after condensate formation and imaged the droplets after an additional hour of incubation. As shown in Figure 4F, the +UV45 samples exhibited the lowest fluorescence intensity compared to both the +UV45_Premix and PT40 samples, with control samples displaying the highest intensity (Figure 4G). Consistent with these observations, FRAP analysis of droplets containing fluorescent components showed rapid DNA fluorescence recovery in both +UV45_Premix and PT40 droplets, but no recovery in +UV45 within the timescale of the experiment (Figure 4H). Finally, Microscale Thermophoresis (MST) performed on these condensate samples revealed significantly reduced mobility in +UV45, consistent with the formation of inter-chain bonds (Figure 4I). Together, these findings support a model in which optically regulated interplay between local or long-range intra-chain and inter-chain interactions governs condensate mechanics.

UV-stabilized droplets exhibit stability and internal compartmentalization
The above observations suggested that UV exposure cross-links DNA molecules in the condensates, thereby altering condensate mechanics and promoting the formation of arrested fusion droplets, which in turn affects chemical compartmentalization. To further assess stability and compartmentalization, we examined the response of +UV45 droplets to extreme changes in the surrounding environment. This was done by replacing the dilute phase with two extreme conditions that, according to our KCl concentration phase diagram, did not support droplet formation. To do this, following UV irradiation, we removed the original dilute phase and replaced it with pure water (0 M KCl) or an aqueous solution of 1 M KCl. In both cases, the condensates remained stable. In the case of 0 M KCl, phase-contrast microscopy and confocal imaging with FITC-labeled PLL (green) and TYE-665-labeled dT40 (red) revealed the formation of dilute regions within the droplets, indicating an electrostatically driven collapse of the dense network57 (darker circles inside the droplets in Figures 5A-D). Washing thus revealed partial compartmentalization induced by UV illumination, which indicates that not all regions were uniformly crosslinked or equally stabilized. Similar dilute compartments have been recently observed in multiple condensate systems upon changes in the ionic strength or temperature57. Erkamp et al. showed that this phenomenon occurs when the equilibrium density of the dense phase changes faster than molecular relaxation within the droplet 57. Specifically, if the droplet contracts faster than the polymer can diffusively redistribute across the droplet, the contraction will open holes of dilute phase within the dense phase. In our system the removal of salt results in tighter association between dT40 and PLL, favoring contraction of the dense phase network, which occurs much faster than the ~10 minute timescale required for molecular redistribution (Figure 4H).

At 1M KCl, confocal fluorescence imaging revealed a marked decrease in overall fluorescence intensity compared to droplets treated with pure water (Figure S5A and B). This reduction indicates a partial release or redistribution of molecular components within the highly screened electrostatic environment. Additionally, unlike the UV-untreated PT40 droplets (Figure S6), fluorescence intensity line scans across individual droplets revealed an interface with high fluorescence intensity (Figure S5 C) in both FITC and TYE channels.

Direct analysis of droplet fusion dynamics using SPM
Our morphological observations indicated that UV-induced thymine dimerization markedly affects droplet fusion dynamics. To directly quantify these effects, we developed an SPM-based assay for condensate droplet fusion and applied it to control and +UV45 samples. Figure 6A presents the experimental setup for this measurement. A dual-coated Petri dish with BSA and Pluronic F127 (PF127), as described in detail in the methodology section, was used for making the condensate system. Droplets on the Pluronic-coated surfaces were less sticky and more spherical compared to the BSA-coated side. This approach allowed us to easily capture a droplet, move to the BSA side to approach the target droplet, and perform a droplet-droplet fusion experiment (Figure 6B).

Our workflow began by selecting a target droplet on the PF127 side and imaging it for diameter determination. The cantilever then approached the droplet under a set force of 0.2 nN (waiting time of 0 seconds), which was sufficient for attachment, thereby capturing the droplet. It was subsequently transferred to the BSA side and positioned adjacent to a selected surface droplet. After imaging, the cantilever’s position was adjusted to align the centers of both droplets, and the cantilever was then advanced toward the surface at a constant (“extend”) velocity of 1 µm s⁻¹ (Figure 6B), while force–time traces were continuously recorded.

Figure 6C shows a typical force–time measurement recorded during the interaction of two liquid droplets. Initially, the force remained near zero, indicating that the droplets were not yet in contact. As the cantilever moved downward, the force rapidly shifted (starting at t1) to a negative (adhesive) regime when the droplets first came into contact (t2), consistent with the formation of a liquid bridge, although direct imaging of the bridge was not possible with our SPM setup. We refer to the time interval between these two events as the liquid bridge formation time (Δt = t2 -t1). After this initial phase, the cantilever continued to move downward at the same speed as the fusion process proceeded to completion. The (liquid) droplets then merged into a single droplet on the substrate. During the subsequent retract phase, the cantilever moved away from the surface, and the force profile remained in the adhesive regime until the connection between the cantilever and merged droplets ultimately broke. Once detached, the cantilever returned “dry” to its original position, leaving an enlarged droplet on the substrate.

Notably, our data revealed a significant difference in the force-time profiles of UV-treated and untreated samples. In non-crosslinked liquid-like droplets, the force drops instantly and then exhibits a two-stage recovery (Figure 6D) after reaching the negative minimum: an initial rapid increase due to fast bridge expansion, followed by a slower phase associated with droplet relaxation and substrate wetting. We observed significantly shorter Δt (Figure 6F) and a higher maximum adhesive force (Figure 6G) in the fusion of control droplets. By contrast, in +UV45 droplets with gel-like behaviour, the force decreases gradually, and the distinct two-regime behaviour is absent; instead, the force recovery occurs gradually and in a continuous manner (Figures 6E, S8).

To examine the geometric scaling expected for capillary coalescence, we compared 𝐹adhesive,max with the reduced radius 𝑅∗ = 𝑅1𝑅2/(𝑅1+𝑅2) for each droplet pair 58. Control droplets followed the linear relation 𝐹adhesive,max = 4𝜋𝛾𝑅∗, yielding an apparent interfacial tension (Fig. 6H and S9). UV-treated droplets systematically deviated below this line, which can be attributed to the viscoelastic contributions, not included in the simple capillary model of pure liquids. The observed deviation from the capillary limit can be rationalized by considering that crosslinking introduces an additional mechanical resistance that reduces effective adhesion. To represent these viscoelastic contributions in a compact phenomenological way, we introduced an empirical modulation factor 𝑆(𝐺′,𝐺′′), yielding
𝐹adh,max ≈ 4𝜋𝛾𝑅∗ / S(G′,G′′). (1)
For un-crosslinked capillary liquids, 𝑆 ≈ 1, whereas in viscoelastic droplets 𝑆 > 1 increases with storage and loss moduli. Several functional forms for 𝑆 reproduce the measured trend, including a rational form
S(G′,G′′) = 1+𝑎 ( 𝐺∗ / 𝐺0 -1), (2)
(with 𝐺* = √𝐺′2 +𝐺′′2) and smooth sigmoidal crossover variants, including a logistic form (Note S2).
While not intended as full mechanical models, these phenomenological descriptions capture the continuous transition from a capillary-dominated (surface tension driven) to a viscoelastic-dominated regime in an internally consistent manner. 𝐺0 represents the corresponding modulus of the un-crosslinked liquid, providing a dimensionless scaling. The approach is most robust under conditions of slow coalescence, a regime that is characteristic of viscoelastic systems59. When applied to other systems, the same framework can be adapted by adjusting parameters to match the characteristic crossover behaviour of different condensate chemistries.

We note that the 𝛾 values calculated from rheology closely agree with those extracted from the capillary model (Figure 6H) for control droplets; however, the measured values are slightly smaller, which can be attributed to mild facilitation of fusion due to favorable molecular interactions in condensates as compared to pure capillary coalescence (accounted for by 𝑆 in the model, see Note S2). Overall, our observations suggest a model in which the gel-like +UV45 droplets exhibit fusion behaviour that is strongly influenced by viscoelasticity (controlling the absolute fusion force), while the heterogeneity in the normalized fusion force reflects contributions from fluctuations in surface tension as well (See Note S3).

Finally, we examined whether the balance between the occurrence of inter- and intra-chain TT dimers affects fusion dynamics. Our rheology analysis showed that in the regime dominated by intra-chain contacts, as seen in +UV45_Premix, both storage and loss moduli are substantially smaller than when inter-chain contacts are frequent, as in the case of +UV45 (Figure S10). Furthermore, surface tension follows a similar trend, with +UV45 droplets exhibiting higher surface tension. Given that 𝜙 = (𝛾Premix × S(UV45)) / (𝛾UV45 × S(Premix)) > 1, the absolute value of the adhesive force is expected to be higher in +UV45_Premix. Consistent with this, our experimental analysis showed that adhesive force is reduced for +UV45, and the slow structural relaxation of its crosslinked network leads to a marked increase in Δt (Figure S10, and 7). Moreover, the decrease in Ψ from Premix to +UV45 (observed in both models; See Note S3) is at least 25%, suggesting that accumulation of inter-chain crosslinks suppresses the sensitivity of fusion forces to viscoelastic fluctuations (consistent with the effective saturation of the moduli), thereby shifting the dominant source of variability toward interfacial mechanics.

Methods
Preparation of ssDNA and Poly-L-Lysine Stock Solutions
ssDNA oligo of dT40 was purchased from Integrated DNA Technologies (NJ, USA). Dry stocks were reconstituted in RNase-free water, without added salt, and DNA concentration was measured subsequently using a Qubit spectrophotometer. The DNA stocks were then aliquoted and stored at -20°C until use. Poly-L-lysine (PLL) hydrochloride with an average molecular weight of 15,000 to 30,000 Da was used in all experiments. The PLL was dissolved in water to prepare stock solutions at 22.4 mg/ml. Based on the average molecular weight, PLL is ~150 residues long, representing a simple model for the diversity of positively charged polypeptides in cells or primitive life forms.

Surface passivation for rheological and droplet-droplet interaction analysis
Condensate samples for phase-contrast imaging, mechanical measurements, and droplet-droplet fusion assays were prepared on TPP Petri dishes. For rheology, dishes were passivated with 1% w/v BSA (A2153) for 30 min and washed five to six times with RNase-free water.

For droplet-droplet fusion, droplets needed to be detached from the substrate with the cantilever and positioned onto target droplets. To enable this, the dish was half-coated with Pluronic and half with BSA: Pluronic provided weak adhesion for easy droplet detachment, whereas BSA promoted stronger adhesion, ensuring stable coalescence during fusion. A guideline was drawn on the underside of the dish to ensure consistent passivation. The dish was fixed in position and tilted ~20°. For each passivation, the bottom half of the dish was filled to the guideline, incubated for 30 min, and washed multiple times with RNase-free water.

Sample preparation
For each experiment, 60 μL of the working buffer (150 mM KCl (p9333), 10 mM imidazole (56750), and 0.01% w/v NaN3 (8.22335))43 was placed as a round droplet at the center of the dish. For droplet-droplet analysis, this step was performed carefully to align the droplet's middle axis with the drawn guideline. To mimic a crowded biological environment, the crowding agent Ficoll® PM 70 (F2878) was added to the buffer at a final concentration of 50 g/L. Subsequently, ssDNA and poly-L-lysine (PLL, P2658) were added sequentially to reach a final concentration of 30 µM for each. After adding each component, the solution was gently mixed by pipetting 10 times.

SPM measurements
A JPK CellHesion 200 (Bruker, Germany) equipped with a phase contrast microscope delivering real-time in-situ images of measurements utilizing a 20X/0.4 objective was employed in this study. The system spans the piconewton to micronewton regime, depending on cantilever stiffness, and provides an extended vertical range of motion, enabling mechanical interrogation of biomolecular condensates across molecular to mesoscale forces. Here, mechanical analysis was performed as explained by Naghilou et al.22 Briefly, a 60 µl bulk drop containing the condensate components was prepared, and the condensate droplets were allowed to settle on the dish. All measurements were performed within 4 h of droplet formation, unless stated otherwise. Before each mechanical measurement, a 5 µl drop was carefully taken from the top of the bulk droplet and placed on the cantilever to avoid the generation of air bubbles when the head is placed. Micrometre-scale indenters were preferred over nano-scale counterparts for improved signal-to-noise ratio44. For all experiments, SAA-SPH-5UM cantilevers (Bruker, Germany) made of Si3N4 with a hemispheric tip (23 µm height, 5.13 µm radius) were used. The calibration of the amplitude and the exact cantilever spring constant was determined with the thermal noise method45. The cantilever was passivated with 1% Pluronic F127 (P2443) for 30 min to prevent the adhesion of droplets on the tip after measurements46. Condensates (preferably of 8- 10 µm in diameter) were indented with a 0.3 nN set force and a cantilever approaching with a constant velocity of 1 µm/s. Acquired data was post-processed by Bruker data analysis software and prepared for calculation of rheological parameters using custom Mathematica code (Mathematica 14.0, Wolfram). Size distribution analysis and calculation of the radius of condensates were done using ImageJ (https://imagej.nih.gov/) from the phase contrast images.

Confocal fluorescence imaging
Confocal fluorescence imaging was performed using a Nikon Ti2 inverted microscope with a Nikon C2plus confocal system and a Plan Apo VC 20x DIC N2 objective (NA = 0.75, WD = 1000 μm). Imaging was conducted with a Galvano scanner in one-way scan mode at 6.897x zoom. Excitation was achieved with 489 nm and 562 nm lasers for GFP and TYE-665, respectively, with emissions detected at 540 nm and 665 nm. The detector gain was set to 100 (GFP) and 70 (TYE-665), and the pinhole size was 40 μm. A brightfield transmission detection (TD) channel was also acquired. Images (512 × 512 pixels) were acquired in three planes: GFP, TYE-665, and TD, with an exposure time of 1095.456 ms at a voxel size of 1×1×1 pixel³. Image processing and analysis were performed using Fiji (ImageJ) version 2.14.0/1. TIFF-formatted images were used for quantitative and structural analysis without additional thresholding or scaling.

UV–visible spectroscopy
UV–visible spectroscopy was performed to evaluate photochemical modifications to the dT40 oligonucleotide following UV irradiation. A final concentration of 30µM dT40 was used for all experiments. For the +UV45_Premix samples, the DNA was mixed into 60µl of working buffer and irradiated for 45 minutes, after which a sample was taken for spectroscopic analysis. For the +UV45_4h samples, 45 minutes of irradiation were applied after 3 hours and 15 minutes of the condensate formation period. Subsequently, high salt concentration was added to the droplets to release the dT40 into the dilute phase, from which a sample was collected. Non-irradiated control samples were prepared by mixing dT40 in the working buffer and analysed immediately. Absorption spectra were recorded using an Agilent 8453 UV–Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) in a low-volume quartz cuvette across the standard nucleic-acid absorption range. The resulting spectral changes were used to identify UV-induced TT dimerization in dT40.

Microfluidic imaging (MFI)
Particle imaging was performed using a ProteinSimple MFI system (ProteinSimple, San Jose, CA, USA) operated in basic mode using default acquisition parameters with a 200 µl/minute flow rate. Particle identification and quantification, including particle counts, equivalent circular diameter (ECD), and circularity, were carried out with the instrument’s analysis software (MFI View System Software, ProteinSimple, San Jose, CA, USA). Processed datasets were exported and plotted using OriginLab 2025 (OriginLab Corporation, Northampton, MA, USA). The approach was applied to +UV45_0h samples, right after UV illumination, and the size and morphology of the floating objects were quantified.

Fluorescence recovery after photobleaching (FRAP)
FRAP experiments were performed to quantify the relative recovery dynamics of PLL and DNA within individual condensate droplets. To enable simultaneous measurement of both components, 5% FITC-labeled PLL (P3543; Sigma-Aldrich, St. Louis, MO, USA) was mixed with its unlabeled counterpart, and 5% TYE-665–labeled dT40 was incorporated into the DNA component. Sample preparation followed the procedures described above.

All measurements were carried out on a Nikon Eclipse Ti2 microscope (Nikon Instruments, Tokyo, Japan) equipped with a 20×/0.75 NA objective. A circular region of interest (radius 1.25 μm) within each droplet was bleached for 1 s using a 488 nm laser at 250 μW, ensuring that droplet diameters were at least two to three times larger than the bleach radius. Fluorescence recovery was recorded for 3 min at low excitation power to minimize additional photobleaching. Both fluorescence channels (FITC for PLL and TYE-665 for DNA) were acquired sequentially for each droplet. Fluorescence intensities were extracted from the bleached region (𝐹ROI), an unbleached reference droplet (𝐹ref), and a background region (𝐹bkgd). To correct for photodecay, intensity traces were normalized to the reference droplet according to:
Fnorm(t) = (FROI(t)−Fbkgd(t)) / (Fref(t)−Fbkgd(t)) ⋅ (𝐹ref,i−𝐹bkgd,i) / (𝐹ROI,i−𝐹bkgd,i) (3)
where 𝐹i denotes the pre-bleach average intensity. Normalized recovery curves were used to compare relative recovery times of PLL and DNA within the same droplets.

Microscale thermophoresis
Microscale thermophoresis (MST) was used to assess the mobility of dT40 molecules in PT40, +UV45_Premix, and +UV45 samples. All samples were prepared at a final dT40 concentration of 30 µM. For the +UV45_Premix condition, dT40 was added to 60 µL of the bulk solution, irradiated for 45 min, and samples were collected immediately after irradiation. For the +UV45 condition, droplets were allowed to form for 3 h 15 min before 45 min of UV exposure. High-salt buffer was then added to dissolve the condensates and release dT40 into the dilute phase, from which material was collected. Non-irradiated controls were prepared by mixing dT40 directly into the working buffer and measured immediately. MST measurements were performed on a Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany) using standard capillaries. Samples were equilibrated for 10 min at room temperature before loading. Fluorescently labeled DNA (5% TYE-665 dT40) was mixed with unlabeled samples. Thermophoretic traces were recorded at 25 °C using medium IR-laser power. Normalized fluorescence (Fnorm) was extracted using the manufacturer’s software, and changes in Fnorm were used to quantify differences in ssDNA mobility associated with UV-induced TT dimer formation.

Anti-CPD immunofluorescence staining assay
Cyclobutane pyrimidine dimer (CPD) formation within condensates was assessed by immunofluorescence staining using a FITC-conjugated anti-CPD antibody from the OxiSelect™ Cellular UV-Induced DNA Damage Staining Kit (CPD, Cell Biolabs). Condensate samples contained 5% TYE-665–labeled DNA (of the total DNA) and were incubated with the anti-CPD primary antibody diluted 1:100 in assay diluent for 1 h at room temperature with gentle agitation. After three washes with 1× wash buffer, droplets were incubated with a FITC-conjugated secondary antibody (1:100 in assay diluent) for 1 h under identical conditions. Excess antibody was removed by four additional washes with 1× wash buffer. Fluorescence microscopy was performed using channels appropriate for both TYE-665 and FITC fluorescence detection.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Sheikhhassani et al. studies how UV-induced photochemical reactions alter the mechanics of ssDNA\ncontaining (PLL/dT40) condensates. The authors found that UV illumination leads to solidification of condensates, likely\nthrough thymine dimerization. They also observed the formation of internal compartments and arrested fusion of\ncondensates.\nOverall, the paper is well organized and easy to read. However, while several observations are interesting, most data are\nqualitative (with the exception of Figure 2) and lack molecular insights. Additionally, the biological relevance of the current\npaper appears limited. Therefore, we believe a properly revised version of the manuscript is better suited for a more\nspecialized journal.\nOur comments are listed below:\n1. The authors used the scanning probe microscopy (SPM) method developed in a previous study. However, comparison\nbetween SPM and existing tools (OT, micropipette aspiration) should be discussed more objectively. Importantly, it’s unclear\nwhy the authors claim SPM to have ‘minimal perturbation’ and ‘more straightforward to integrate with UV illumination’\ncompared to other assays.\n2. In SPM measurements, it’s unclear if passivation is necessary and if it will affect the viscoelasticity quantifications.\n3. It’s important to provide the power density of the UV illumination.\n4. The extensive use of condensate morphology (eg, circularity, semi-fusion, elongated shape) is concerning. This is\nbecause A), the morphology of condensates are strongly affected by the adhesion between condensates and the substrate\n(this adhesion is clearly not removed for most experiments). For example, see (https://www.freeimages.com/premium/water-\ndroplets-on-a-metal-surface-1592483 ). B) condensates with similar circularity can have very different mechanical properties,\nas illustrated in several recent studies (for example, https://pubmed.ncbi.nlm.nih.gov/39211102/ ).\n5. The authors should better explain the size distribution of condensates and their corresponding formation mechanisms.\nThese are unlikely common knowledge for the readers. Along this line, how does condensate surface tension affect its size\ndistribution?\n6. In figure 2, the observation that the crossing point between G’ and G’’ increase between 30 and 45 min UV is surprising.\nHow does this reconcile with the solidification of the condensate and the observed change in terminal viscosity (which itself\nneeds to be better defined)?\n7. Does aging play a role in the liquid to solid transition of the PT40 condensates? For example, it would be better to include\na set of data a separate plot to show the material properties of PT40 is stable for at least 45 min.\n8. A major point of the paper is thymine dimerization induced by UV. While plausible, we didn’t find any direct evidence in\nthe paper to support this claim. Could the authors check with electrophoresis?\n9. The observation of compartments within condensates is interesting. But this result is very descriptive and lack of\nmechanistic insights.\n10. The fusion between two condensates is affected by condensate viscosity. So, it’s unclear if data in figure 5 can be used\nto draw direct conclusion on condensate surface tension and if it is affected by the speed of the measurement. Plus, the\nclaim of ‘bridge formation’ does not appear to be directly supported by imaging evidence.\nMinor problems:\n1. Line 82 ‘preserving chemical composition’ is inaccurate since dimerization is a chemical change.\n2. In line 167, the author mentioned “this condition produced droplets of convenient size for mechanical analysis” but did not\nstate what is the actual size in the main text or main figures.\n3. Figure 1A and many other images are missing scale bar.\n4. Figure 1F. I would recommend defining what is single/paired/cluster with images.\n5. Line 232, unclear if the values were quoted for a specific frequency.\n6. Line 246, reference missing.\n7. The description and alignment of Figure 3A and 3B are overall confusing. Since A and B are two different experiments. It\nis better to make separate text descriptions for them under figure 3.\n8. Although “applying UV immediately after mixing (+UV45_0h)” was observed with no condensate formation in 3A, it still\nrecommended to include a confocal image of +UV45_0h with 1% TYE-665 tagged dT40 in Figure 3B.\n9. Figure 3F meant to show no significant differences between control and premix samples but the control is not plotted.\n10. Line 394, the use of reduced radius is unclear.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "In the submitted manuscript, scanning probe microscopy is combined\nwith optical microscopy to study the mechanical properties and fusion\ndynamics of condensate droplets. The droplets are formed in solutions\nof two polyelectrolytes, single-stranded DNA consisting of 40 thymine\nnucleotides (oligo-ssDNA dT40), which carries 40 negative charges,\nand the protein PLL with an average length of 150 residues, carrying\n150 positive charges. The solution undergoes phase separation within\na certain concentration regime as characterized in Figure S1.\nWhen the polyelectrolyte solution is illuminated with UV light, the two\npolymers form crosslinks which can drive the droplets from a liquid to\na gel-like or solid-like state, thereby changing the mechanical properties\nof the droplets and their morphology. The transition from the liquid to a\ngel- or solid-like state can be used, for instance, to arrest the fusion of\nthe condensate droplets in some intermediate droplet state with\nnonspherical morphology as shown in Figure 1G.\nMy main concern is that the results reported in this manuscript are\nquite reasonable from an intuitive and qualitative point of view whereas\nthe analysis of these results is rather fragmented, often confusing, and\ndifficult to follow.\nI recommend major revision of the submitted manuscript. This revision\nshould, in particular, address the following points, ordered in the same\nway as the corresponding manuscript pieces in the main text and in\nthe supplementary information:\n1) The title of the manuscript emphasizes thymine dimerization and\nthe abstract claims on page 1, line 27 and 28, that \"UV-induced\nthymine dimerization alters condensate nucleation and coalescence\",\nHowever, this claim remains obscure until Figure 6 at the end of the\nmanuscript. Therefore, I urge the authors to mention the competition\nbetween covalent intra- and interchain contacts, which is currently\nexplained in Figure 6, already in the abstract and to further clarify\nthe text pieces before and after Figure 6.\n2) At the end of the abstract, the authors claim that their findings\nhave \"implications for genome biology, the emergence of life on\nEarth, and engineering applications.\"\nI can see possible engineering applications, related, for instance,\nto synthetic biology but I have severe problems to imagine\nimplications for genome biology and for the emergence of life on\nEarth. In fact, \"genome biology\" is not mentioned anywhere else\nin the manuscript and the \"origin of life\" is only briefly mentioned\nin line 446 on page 17 at the very end of the manuscript.\nIf the authors want to maintain these claims, they should tell the\ninterested reader in much more detail what they have in mind.\n3) Related to point 2): Condensate droplets in living cells have been\ndiscovered in 2009 and have been intensely studied during the last\ntwo decades by a variety of experimental and theoretical methods.\nIt is now understood that intracellular condensate droplets are\nusually in a liquid-like rather than in a gel-like or solid-like state.\nTherefore, do the authors really believe that gel-like or solid-like\ncondensate droplets play some important role in living cells?\nIf yes, they should explicitly describe their line of reasoning in the\nmanuscript.\n4) The first paragraph of \"Materials and Methods\" provides some\ninformation about the preparation of the stock solutions. This\ninformation is, however, not sufficient if someone wants to repeat\nthis preparation.\nAs far as dT40 is concerned, one would like to know about the\nbuffer used to rehydrate the dry dT40 obtained from the producer.\nIn particular, which salt was added at what concentration? And\nwas the pH controlled during this preparation?\nAs far as the PLL protein is concerned, I would have expected\nthat this protein was produced by some recombinant method,\nwhich would lead to a well-defined length of the protein.\nHowever, in line 101 and 102 on page 4, the authors mention\nthe \"average\" molecular weight and the \"average\" length of the\nPLL protein. Where does the polydispersity of PPL come from\nand how large is it?\n5) After cross-linking the two polymers by UV illumination, the\ndroplets exhibit an increased viscosity as reported in Figure 2E.\nThis increase is dramatically different depending on the protocol\nused for the illumination. The lettering at the x-axis of Figure 2E\ndistinguishes three cases: \"control\", \"30minUV:4hrs\", and\n\"45minUV:4hrs\".\nOne might think that \"xyminUV:4hs\" stands for \"applying UV for\nxy min after 4 hours\", but these abbreviations are not explained\nin the text. Instead, in line 241 on page 9, the same protocols\nare called \"+UV30\" and \"+UV45\". Furthermore, in line 249 on\npage 9, the authors say that the UV irradiation was applied \"for\n45 minutes\" without mentioning the alternative exposure time\nof 30 minutes. In addition, the authors now refer to the \"Premix\"\ncondition and to the \"0 hours\" condition.\nTherefore, the description of the different protocols as used in\nthe submitted manuscript is very confusing and must be\nimproved. The same comment applies to Figure 3.\n6) Related to point 5): One general advantage of light-induced\nchanges of molecules and chemical reactions is that these\nchanges can be rather fast. Indeed, the publication by\nJohnson and Wiest, JPC B (2007), Ref 11 of the manuscript,\nemphasizes that \"thymine dimerization is an extraordinarily\nrapid reaction.\" In contrast, the time scales discussed in the\nsubmitted manuscript for the UV illumination protocols are\nvery long, corresponding to 30 or 45 minutes.\nThus, the authors should explain why the UV-induced\ndimerization studied here is so much slower.\n7) The results for the apparent interfacial tension are displayed\nin Figure 5H in the form of a histogram. This histogram exhibits\nat least two, maybe even three peaks. The authors should explain\nwhether or not they can interpret this histogram in terms of the\nunderlying molecular processes.\nDoes this histogram reflect, for instance, the competition between\ncovalent intra- and interchain contacts of the polymer chains?\n8) Two typos on page 15: in line 399, Figure 5H should be replaced\nby Figure 5G, and in line 402, Figure 5I should be replaced by\nFigure 5H.\n9) Figure S1 is quite useful in order to understand the formation\nof the biomolecular condensates studied here, Therefore, the\nauthors might want to move Figure S1 into the main text.", "reviewer_4": "In a nutshell, the paper reports that UV illumination can alter the fusion times of condensate droplets. I haven’t studied the\nliterature exhaustively, but this does seem to me like a new and interesting discovery. I have comments:\n• I am not an expert in this specific area, but the authors state with great confidence that the observed changes in\ncondensate mechanics are due to thymine dimerization. I do not see direct evidence for this claim; at present it seems more\nlike a plausible hypothesis rather than a demonstrated molecular mechanism.\n• In Fig. 1G and H the authors analyze “neck length.” While this is a reasonable empirical observation, is there a quantitative\nframework that could connect this parameter to rheological or other mechanical quantities?\n• I find the introduction poorly written and the results written with a somewhat overstated style. For example:\no Micropipette aspiration does not, in general, require the use of tracer particles.\no To my knowledge, thymine dimerization involves the formation of covalent bonds, which necessarily alters the chemical\ncomposition. Therefore, the sentence claiming that chemical composition remains unchanged should be revised.\no The text contains multiple “first-of-its-kind” statements that are unnecessary.\no Prior analogous work should be acknowledged, for instance the use of AFM to measure the material properties of objects\nas large as condensates (https://www.nature.com/articles/nphys4104).\no The paper is about material properties and their relation to molecular conformations, but does not cite the significant very\nrecent progress in this area from the Banerjee, Mittal, and Schuler groups.\n• Line 332 seems to be referring to Figure 5B. Please check.\n• Finally, I find it puzzling that no condensates are reported in the “0h” condition. Could it be that phase separation does\noccur, but the droplets are arrested at sizes below the diffraction limit of the imaging system?"}, "author_response": "We are grateful to the referees for their critical feedback and suggestions, which helped\nus improve the manuscript. Below, we provide point-by-point responses to their\ncomments and describe the revisions made.\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Sheikhhassani et al. studies how UV-induced photochemical\nreactions alter the mechanics of ssDNA containing (PLL/dT40) condensates. The authors\nfound that UV illumination leads to solidification of condensates, likely\nthrough thymine dimerization. They also observed the formation of internal compartments and arrested\nfusion of condensates.\nOverall, the paper is well organized and easy to read. However, while several\nobservations are interesting, most data are qualitative (with the exception of Figure 2)\nand lack molecular insights. Additionally, the biological relevance of the current paper\nappears limited. Therefore, we believe a properly revised version of the manuscript is\nbetter suited for a more specialized journal.\nResponse:\nThanks for your positive remarks on the structure and readability of the paper and the\nconstructive comments. As described below, we made extensive revisions to the paper\nand added substantial new data to better highlight the findings and impact of our work\non the field of biomolecular condensates. Importantly, we have addressed the reviewer’s\nconcern regarding the qualitative nature of the data and underlying molecular\nmechanisms, which we elaborate on in the remainder of this rebuttal. The work reveals\nhow the balance of inter- versus intrachain contacts governs condensate mechanics and\nfusion, providing key insights into biomolecular phase behavior.\nOur work presents unprecedented insights into the control and engineering of\ncondensate microdroplets and introduces powerful tools and model systems for\ninvestigating condensate mechanochemistry. These technological innovations enabled\nus to make the following key discoveries:\n1) Chemistry affects condensation: Our study shows that the simplest chemical\nreaction involving a photochemical-induced formation of a covalent bond affects\ncondensation without changing the chemical content of the system.\n2) Condensation affects chemistry: We show that the outcome of a\nphotochemical reaction is tightly dependent on the presence or absence of\ncondensation.\nMoreover, the demonstrated ability to control condensate phase and\nmechanochemistry, as well as to synthesize compartmentalized condensate particles,\nprovides new bio-inspired engineering approaches relevant to chemical engineering and\nmaterials science.\nTherefore, we believe that our study offers fundamental insights into the relationship\nbetween condensation and chemical reactivity, while also introducing unique, versatile\ntools for the precise engineering of condensate properties. This work establishes\nbiomolecular condensates as a chemically programmable system, linking their\nmechanochemistry to reaction control and material design, with broad implications for\nchemistry beyond biology. While we have revised the manuscript to avoid overstating\nimmediate biological relevance, we emphasize that the technological contributions are\nexpected to have a significant impact on both chemical and biological research. Given\nits broad implications, we are of the opinion that our work will be of great interest to the\nreaders of Nature Communications.\nOur comments are listed below:\n1. The authors used the scanning probe microscopy (SPM) method developed in a\nprevious study. However, comparison between SPM and existing tools (OT, micropipette\naspiration) should be discussed more objectively. Importantly, it’s unclear why the\nauthors claim SPM to have ‘minimal perturbation’ and ‘more straightforward to integrate\nwith UV illumination’ compared to other assays.\nResponse:\nThanks for this comment. In the original version, our description of SPM included\nstatements such as “minimal perturbation” and “more straightforward to integrate with\nUV illumination,” which were unnecessary and could be interpreted as overclaims. We\nhave removed these phrases and rewritten the section to avoid implying advantages that\nare not essential to our argument. SPM does provide a convenient way of performing\nthese experiments due to the easy integration of a UV treatment module. While such\nintegration is theoretically possible with custom-built optical tweezers, current\ncommercial systems do not offer a practical way to achieve this.\nWe have fully rewritten the corresponding introductory paragraph and added references\nto our recent studies, published while this manuscript was under review, where we\nestablished the use of SPM for condensate rheology analysis. In these papers,\ncomparisons with other techniques are discussed objectively, including the relevant\nforce regimes and whether tracer particles or exogenous probes are required.\nFinally, we emphasize in the revision that the use of SPM for direct droplet–droplet fusion\nmeasurements is new to this work. In contrast to optical tweezers, where force\ncalibration is challenging in condensate fusion studies, SPM allows straightforward\ncalibration. Moreover, the open geometry and ability to mechanically manipulate\ndroplets make it feasible to bring separately treated droplets into controlled contact and\nsubsequently characterize the merged object capabilities that are difficult to implement\nin closed OT chambers. Specific droplets can then be subjected to downstream analysis\nusing analytical methods.\n2. In SPM measurements, it’s unclear if passivation is necessary and if it will affect\nthe viscoelasticity quantifications.\nResponse:\nThanks for this comment.\nPassivation is indeed necessary for SPM analysis of these condensates to ensure\nthe formation of round droplets. Making condensates on uncoated SPM dishes\ntypically leads to the spreading of the condensates due to wetting. The reviewer\nasks a valid question on whether passivation has any impact on viscoelasticity\nquantifications. While passivation is commonly used in biomolecular condensate\nresearch, we agree that this aspect merits careful investigation. To address this,\nwe performed new sets of experiments in which two very distinct passivation\nprotocols were used, BSA and PF127, and then subjected the samples to\nrheological analysis on round droplets. Our findings demonstrate that, for our\nsystem, the impact of passivation on condensate rheology is small compared to\nthe TT dimerization-induced viscoelastic changes that we are investigating.\nFigure 1. Rheological analysis of PT40 samples on BSA and PF127 passivated surfaces.\n3. It’s important to provide the power density of the UV illumination.\nResponse:\nThanks for this comment. In response to this comment, we measured the power density\nand provided the results in the revised manuscript.\n4. The extensive use of condensate morphology (eg, circularity, semi-fusion,\nelongated shape) is concerning. This is because A), the morphology of\ncondensates are strongly affected by the adhesion between condensates and the\nsubstrate (this adhesion is clearly not removed for most experiments). For\nexample, see (https://www.freeimages.com/premium/water-droplets-on-a-metal-surface-1592483 ). B) condensates with similar circularity can have very different mechanical properties,\nas illustrated in several recent studies (for example, https://pubmed.ncbi.nlm.nih.gov/39211102/ ).\nResponse:\nThanks for this comment. While we agree that surface properties impact the\nmorphology of condensate droplets, our comparative analysis is focused on UV-\ninduced changes under constant surface chemistry. As such, the morphological\nchanges can be attributed to DNA-induced thymine dimerization within the\ncondensates. To better demonstrate this, we performed new experiments where\nwe generated condensates on two different chemistries, with high and low\nadhesiveness, and subjected the condensates to UV illumination. The UV-\ninduced morphological changes are apparent and can be distinguished from\nsurface effects. More specifically, we performed the UV irradiation experiment on\nsurfaces passivated with Pluronic F-127 (PF127), a coating known to produce\nnon-sticky droplets (as shown in the current work and consistent with Figure 6A)\n(See Yao, et al., PNAS 121(22), (2024)). Following 45 minutes of UV irradiation on\nthese PF127-passivated dishes, we observed the formation of fusion-arrested\ndroplets with significant morphological changes, as we observed on the BSA-\npassivated surfaces. This evidence supports the conclusion that the\nmorphological changes and the formation of doublets are directly attributable to\nthe UV-induced thymine dimerization within the condensates and the associated\nchanges in material properties. We have included these data in a new\nSupplementary Figure S2, which shows consistency with Figure 2B.\nFinally, we do agree with the referee that making a causative link between\nmorphology and mechanics is not straightforward in general. We rephrased the\ncorresponding parts in our manuscript to avoid confusion. In our study, however,\nwe do see morphological and mechanical changes upon UV illumination, and the\ncrosslinking of polymers has also been previously reported to affect mechanics\nand morphology in other condensate chemistries (See e.g., Doi:\n10.1016/j.xcrp.2025.102430)\n5. The authors should better explain the size distribution of condensates and their\ncorresponding formation mechanisms. These are unlikely common knowledge\nfor the readers. Along this line, how does condensate surface tension affect its\nsize distribution?\nResponse:\nThanks for this comment. We do agree that we have been too short in our\ndescription, which was due to the journal’s article length limit. In the revised\nmanuscript, we elaborated on size distribution analysis and formation\nmechanics. Given the space limitation, we added this largely to the description of\nFigure S2.\nTo address the comment on surface tension, we measured surface tension using\nSPM for both +UV45 and PT40 droplets and included them in the revised article.\nUV-induced thymine dimerization alters both the surface tension and the\nrheology of the condensates, leading to dynamic changes in droplet size over\ntime. An increase in surface tension promotes coalescence, contributing to the\ngrowth of large droplets, while intermediate-size droplets decrease as they merge\ninto larger structures. By 45 minutes, very large, fusion-arrested droplets appear,\nreflecting rheology-mediated arrest of coalescence, while the smallest droplets\ncontinue to form via fragmentation during incomplete coalescence events.\nMoreover, because illumination begins earlier in the +UV45 assay than in +UV30,\na larger population of small pre-existing droplets becomes crosslinked and\npreserved in +UV45, contributing to the increase observed in the smallest size bin\nrelative to +UV30. This overall pattern is clearly reflected in the size distributions\npresented in the manuscript.\n6. In figure 2, the observation that the crossing point between G’ and G’’ increase\nbetween 30 and 45 min UV is surprising. How does this reconcile with the\nsolidification of the condensate and the observed change in terminal viscosity\n(which itself needs to be better defined)?\nResponse:\nThanks for this comment. Upon moderate UV exposure, G’ and G’’ increase\ndramatically and a crossover appears, indicating a transition from liquid-like to\nsolid-like behavior. At higher UV doses, both G’ and G’’ continue to increase, but\nthe crossover shifts to higher frequencies. This shift arises from the formation of\na heterogeneous network with locally crosslinked domains, which increases\nshort-time stiffness and allows faster relaxation within these smaller domains.\nThe condensate becomes stiffer overall, yet the network relaxes faster at the\ntimescales probed, because motion is confined to these local regions. Unlike a\nfully percolated network characteristic of a complete liquid-to-solid transition,\nthis state represents a partial liquid-to-solid transition, or incipient gelation. The\nshift of the crossover to higher frequency reflects a shorter dominant relaxation\ntime, which falls outside the measurement window. We added an explanation\nabout this in the revised article.\nWe revised the part on terminal viscosity to ensure that its definition and the\nprocedure behind its derivation is clearly stated.\n7. Does aging play a role in the liquid to solid transition of the PT40 condensates?\nFor example, it would be better to include a set of data a separate plot to show the\nmaterial properties of PT40 is stable for at least 45 min.\nResponse:\nThanks for this comment. To address this question, we performed new\nexperiments which reveal that the aging effect is negligible within the\naforementioned time scales, as can be seen from the figure below. We added a\nphrase to the revised paper on this point.\n1000 1000\n100 100\n10 10\n1 1\n10 100\nFigure 2. Rheological analysis of PT40 samples 45 minutes aged after 4 hours of standard incubation time.\n8. A major point of the paper is thymine dimerization induced by UV. While\nplausible, we didn’t find any direct evidence in the paper to support this claim.\nCould the authors check with electrophoresis?\nResponse:\nThanks for this comment. To address this question, we performed the requested\nelectrophoretic mobility check, as well as two other complementary studies. To\nperform this study, we released the DNA from the condensates by introducing salt\nand screening the charged interactions. The droplets got dissolved, and the\nsolution was then subjected to MST (See New Figure 4I). The results clearly show\nthat +UV45 DNAs are diffusing significantly slower than the control, indicating a\nlarger molecular size and crosslinking. UV treatment in Premix condition led to\nexclusively intrachain TT bonds, which leads to reduced gyration radii and higher\nmobility. As another way of proving TT dimer formation, we performed UV\nspectroscopy, which clearly shows formation of TT bonds in agreement with\nprevious reports (New Figure 1H). Finally, we set up an anti-TT dimer antibody\nassay, which showed clearly staining of UV-treated droplets and lack of staining\nof control ones (New Figure 1I). These new studies provide direct evidence for the\nformation of the TT dimer in our samples. We included these new results in the\nrevised article.\n9. The observation of compartments within condensates is interesting. But this\nresult is very descriptive and lack of mechanistic insights.\nResponse:\nThanks for this comment. The compartmentalization phenomenon we observe is\nidentical to that reported by Erkamp et al. (Ref 56). In that work, it was determined\n)aP(\n'G\n)aP(\n''G\nG'', G'' (Aged)\nG', G' (Aged)\nFrequency (Hz)\nacross numerous systems that internal cavities formed when the environmental\nconditions were changed such that the condensate favored a more\ncompact/dense state. If the condensate contraction occurred faster than the\nmolecules could redistribute, the rapid contraction would create cavities of dilute\nphase within the dense droplets. The study of Erkamp et al. used temperature\ndrops and salt additions to reduce the repulsion between like-charged\ncomponents (reducing the temperature causes a decrease in the Donnan\npressure of the neutralizing counterions). Given the strong similarities between\nour system and Erkamp’s, we are confident that we are observing the same\nphenomenon. There are two mechanistic details, however, that differ in our\nsystem. First, the removal of salt in our system promotes the association of\noppositely charged components. Second, the presence of UV-induced crosslinks\nwill lead to much longer redistribution timescales compared to the reptation-\nlimited timescales of Erkamp et al. We have extended the discussion of this\nphenomenon in the manuscript to strengthen the connection with reference 56.\n10. The fusion between two condensates is affected by condensate viscosity. So,\nit’s unclear if data in figure 5 can be used to draw direct conclusion on condensate\nsurface tension and if it is affected by the speed of the measurement. Plus, the\nclaim of ‘bridge formation’ does not appear to be directly supported by imaging\nevidence.\nResponse:\nThanks for this comment. Extend speed does affect the maximum adhesive force\n(as shown in the new data provided in Figure S7. In our study, we operate under\nconstant speed, and the changes in fusion dynamics are due to surface effects,\ngeometry, and rheological properties. To investigate these effects and their\ncontributions, we performed new analyses and developed new models, which are\npresented in the revised article (See Section on SPM analysis of droplet fusion\ndynamics, as well as Supplementary Notes S2 and S3). We measured surface\ntension using an SPM-based approach as well as using the capillary model for the\ncase of control droplets. In +UV45 droplets, the relation between maximum\nadhesive force and surface tension deviates from the capillary models and can be\nexplained by the models that are adjusted for viscoelastic effects.\nRegarding bridge formation: our setup does not have side imaging, and thus, it is\nnot possible to provide imaging evidence. As such, we revised our text to\nacknowledge this point.\nMinor problems:\n1. Line 82 ‘preserving chemical composition’ is inaccurate since dimerization is a\nchemical change.\nResponse:\nThanks for this comment. Of course, we agree that addition or rearrangement of\nchemical bonds is indeed a chemical change. Our intention in using the phrase\n“preserving chemical composition” was to distinguish our approach from more\ncommon strategies in which condensates are chemically modified through the\nintroduction of new molecules or crosslinkers, leading to changes in\ncomposition. In our study, no additional molecules or crosslinkers are introduced\ninto the condensate, thus “atomic (or elemental) composition” is preserved, and\nchemical composition changes are limited to covalent bond formations\n(connectivity). We have revised the text to clarify this distinction while still\nconveying our intended point more accurately.\nRevised introductory phrase reads:\n“…. while preserving chemical composition —that is, without adding new\nmolecules and without changing atomic composition.”\nRevised phrase in the discussion reads:\n“… light can be leveraged to program the connectivity and internal organization\nof a nucleic acid-based condensate system, thereby changing condensate\nmechanics with no alterations to atomic (elemental) composition.”\n2. In line 167, the author mentioned “this condition produced droplets of\nconvenient size for mechanical analysis” but did not state what is the actual size\nin the main text or main figures.\nResponse:\nThanks for this comment. We revised this phrase, and the size information is\nincluded in the manuscript (methods section) and will be deposited in the\nassociated raw data as well.\n3. Figure 1A and many other images are missing scale bar.\nResponse:\nThanks for this comment. We added the missing scale bars.\n4. Figure 1F. I would recommend defining what is single/paired/cluster with\nimages.\nResponse:\nThanks for this comment. This is now included in the revised version, in Figure 2A.\nPlease note that in the revision, we refrained from using two interchangeable\nterms, doublet and paired. All such structures are now referred to as doublets.\n5. Line 232, unclear if the values were quoted for a specific frequency.\nResponse:\nThanks for this comment. We fixed this issue in the revised version.\n6. Line 246, reference missing.\nResponse:\nThanks for this comment. We added a reference.\n7. The description and alignment of Figure 3A and 3B are overall confusing. Since\nA and B are two different experiments. It is better to make separate text\ndescriptions for them under figure 3.\nResponse:\nThanks for this comment. We fixed this issue in the revised version, and fully\nseparated the two descriptions and figure panel arrangements. Please see the\nnew Figure 4.\n8. Although “applying UV immediately after mixing (+UV45_0h)” was observed\nwith no condensate formation in 3A, it still recommended to include a confocal\nimage of +UV45_0h with 1% TYE-665 tagged dT40 in Figure 3B.\nResponse:\nThanks for this comment. We performed new confocal imaging as well as MFI\nanalysis, which are included in Figure S4.\n9. Figure 3F meant to show no significant differences between control and premix\nsamples but the control is not plotted.\nResponse:\nThanks for this comment. We included the control data in the plot, showing that\nthe differences are relatively minimal.\n10. Line 394, the use of reduced radius is unclear.\nResponse:\nThanks for this comment. We checked this part and ensured that the definitions\nand approach are clearly stated.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nThank you for your time and constructive feedback, which helped us improve the\nmanuscript. We have extensively revised the article, incorporating new experimental\ndata, theoretical models, and additional mechanistic insights that not only strengthen\nthe study but also highlight its broad significance and potential impact across chemistry,\nmaterials science, and biomolecular condensate research, consistent with the scope\nand readership of Nature Communications.\nReviewer #3 (Remarks to the Author):\nIn the submitted manuscript, scanning probe microscopy is combined\nwith optical microscopy to study the mechanical properties and fusion\ndynamics of condensate droplets. The droplets are formed in solutions\nof two polyelectrolytes, single-stranded DNA consisting of 40 thymine\nnucleotides (oligo-ssDNA dT40), which carries 40 negative charges,\nand the protein PLL with an average length of 150 residues, carrying\n150 positive charges. The solution undergoes phase separation within\na certain concentration regime as characterized in Figure S1.\nWhen the polyelectrolyte solution is illuminated with UV light, the two\npolymers form crosslinks which can drive the droplets from a liquid to\na gel-like or solid-like state, thereby changing the mechanical properties\nof the droplets and their morphology. The transition from the liquid to a\ngel- or solid-like state can be used, for instance, to arrest the fusion of\nthe condensate droplets in some intermediate droplet state with\nnonspherical morphology as shown in Figure 1G.\nMy main concern is that the results reported in this manuscript are\nquite reasonable from an intuitive and qualitative point of view whereas\nthe analysis of these results is rather fragmented, often confusing, and\ndifficult to follow.\nI recommend major revision of the submitted manuscript. This revision\nshould, in particular, address the following points, ordered in the same\nway as the corresponding manuscript pieces in the main text and in\nthe supplementary information:\nResponse:\nThanks a lot for the critical assessment of the paper and positive remarks on the\nfindings. We carefully studied the comments and did our best to address them all.\nThe paper is also restructured and rewritten in numerous parts to improve\nreadability and flow. In what follows, we describe the revisions we did to the\nmanuscript:\n1) The title of the manuscript emphasizes thymine dimerization and\nthe abstract claims on page 1, line 27 and 28, that \"UV-induced\nthymine dimerization alters condensate nucleation and coalescence\",\nHowever, this claim remains obscure until Figure 6 at the end of the\nmanuscript. Therefore, I urge the authors to mention the competition\nbetween covalent intra- and interchain contacts, which is currently\nexplained in Figure 6, already in the abstract and to further clarify\nthe text pieces before and after Figure 6.\nResponse:\nThank you for this comment. We have revised the abstract to explicitly mention\nthe competition between intra- and inter-chain contacts. In addition, we have\nreworked the text associated with Figure 6 (now Figure 7) and the sections before\nit. The concept of balance between inter- and intra-chain contacts now appears\nconsistently throughout the paper. We also include substantial new experimental\ndata and theoretical results to strengthen this point. In particular, we added a new\nFigure S11, which complements the model presented in Figure 7.\n2) At the end of the abstract, the authors claim that their findings\nhave \"implications for genome biology, the emergence of life on\nEarth, and engineering applications.\" I can see possible engineering\napplications, related, for instance,\nto synthetic biology but I have severe problems to imagine\nimplications for genome biology and for the emergence of life on\nEarth. In fact, \"genome biology\" is not mentioned anywhere else\nin the manuscript and the \"origin of life\" is only briefly mentioned\nin line 446 on page 17 at the very end of the manuscript.\nIf the authors want to maintain these claims, they should tell the\ninterested reader in much more detail what they have in mind.\nResponse:\nThank you for this comment. We agree that our original descriptions were too brief\nand somewhat overstated. Our study is primarily focused on synthetic biology\nand materials science, with some discussion of possible implications for the\norigin of life. Indeed, UV-induced dimerization is a pathological process and\ntherefore has no role in genome physiology; consequently, our reference to\ngenome biology did not accurately reflect this point. In the revised manuscript,\nwe have removed the reference to genome biology and revised the text throughout\nto elaborate on these broader points, while remaining within the journal’s length\nlimits.\n3) Related to point 2): Condensate droplets in living cells have been\ndiscovered in 2009 and have been intensely studied during the last\ntwo decades by a variety of experimental and theoretical methods.\nIt is now understood that intracellular condensate droplets are\nusually in a liquid-like rather than in a gel-like or solid-like state.\nTherefore, do the authors really believe that gel-like or solid-like\ncondensate droplets play some important role in living cells? If\nyes, they should explicitly describe their line of reasoning in the\nmanuscript.\nResponse:\nThank you for this comment. We agree that condensates identified in healthy\nhuman cells are typically liquid-like. Gel-like condensates are frequently\nobserved in pathologies and aging processes. The origin-of-life aspect of our\nstudy considers primitive life forms, and there is evidence that condensates in\nbacteria can adopt gel-like states. We have elaborated on these points in the\nrevised manuscript (and included supporting citations) to avoid potential\nconfusion.\n4) The first paragraph of \"Materials and Methods\" provides some\ninformation about the preparation of the stock solutions. This\ninformation is, however, not sufficient if someone wants to repeat\nthis preparation.\nAs far as dT40 is concerned, one would like to know about the\nbuffer used to rehydrate the dry dT40 obtained from the producer.\nIn particular, which salt was added at what concentration? And\nwas the pH controlled during this preparation?\nAs far as the PLL protein is concerned, I would have expected\nthat this protein was produced by some recombinant method,\nwhich would lead to a well-defined length of the protein.\nHowever, in line 101 and 102 on page 4, the authors mention\nthe \"average\" molecular weight and the \"average\" length of the\nPLL protein. Where does the polydispersity of PPL come from\nand how large is it?\nResponse:\nThank you for this comment. We agree with the referee and apologize for the\nbrevity in our original descriptions. In the revised manuscript, we have included\npractical details, such as the buffer composition, salt concentration, and pH\ncontrol during the rehydration of dT40, to ensure reproducibility of the study.\nWe have also clarified our description of PLL. We intentionally chose to use PLL\nwith a range of lengths as a simple way to model the diversity of positively charged\npolypeptides found in cells or in primitive life forms. Please note that the polymer\nexhibits a distribution of lengths due to its synthesis protocol. Fully defined-length\nPLL can be obtained through synthetic routes, but such approaches are typically\nlimited to short chains and small quantities, not feasible for condensate studies.\nWe clarified these points in the revised version of the manuscript (See Materials\nand Methods).\n5) After cross-linking the two polymers by UV illumination, the\ndroplets exhibit an increased viscosity as reported in Figure 2E.\nThis increase is dramatically different depending on the protocol\nused for the illumination. The lettering at the x-axis of Figure 2E\ndistinguishes three cases: \"control\", \"30minUV:4hrs\", and\n\"45minUV:4hrs\".\nOne might think that \"xyminUV:4hs\" stands for \"applying UV for\nxy min after 4 hours\", but these abbreviations are not explained\nin the text. Instead, in line 241 on page 9, the same protocols\nare called \"+UV30\" and \"+UV45\". Furthermore, in line 249 on\npage 9, the authors say that the UV irradiation was applied \"for\n45 minutes\" without mentioning the alternative exposure time\nof 30 minutes. In addition, the authors now refer to the \"Premix\"\ncondition and to the \"0 hours\" condition.\nTherefore, the description of the different protocols as used in\nthe submitted manuscript is very confusing and must be\nimproved. The same comment applies to Figure 3.\nResponse:\nThank you for this comment and for bringing these issues to our attention. We\nrevised our abbreviation/terminology to avoid confusion.\n6) Related to point 5): One general advantage of light-induced\nchanges of molecules and chemical reactions is that these\nchanges can be rather fast. Indeed, the publication by\nJohnson and Wiest, JPC B (2007), Ref 11 of the manuscript,\nemphasizes that \"thymine dimerization is an extraordinarily\nrapid reaction.\" In contrast, the time scales discussed in the\nsubmitted manuscript for the UV illumination protocols are\nvery long, corresponding to 30 or 45 minutes.\nThus, the authors should explain why the UV-induced\ndimerization studied here is so much slower.\nResponse:\nThank you for this insightful comment. We agree that thymine dimerization is\nintrinsically very fast, as noted in Johnson and Wiest (J. Phys. Chem. B, 2007, Ref\n25). For dimerization to occur, the bases must rotate into the correct orientation\nto form a cyclobutane dimer. For intermolecular dimerization, there is an\nadditional limitation coming from the time required for the molecules to diffuse\ntogether. Such intermolecular reactions were studied by Nagpal et al. (Ref 49),\nwho found that the reaction reached saturation on a timescale on the order of 20\nminutes. These results are not directly applicable to our system for two reasons.\nFirst, the thymine monomers in the Nagpal experiments were at much lower\nconcentrations than the interior of our condensates. Second, their system did not\ninclude phosphate backbones and, therefore, did not exhibit the strong\nelectrostatic repulsion between nucleic acid chains. However, these two effects\nmodify the dimerization rate in opposite directions; reduced concentration\ndecreases the rate, and the lack of phosphates increases the rate relative to our\nsystem. Therefore, our empirical observation that UV continues to affect\ncondensate properties past 30 minutes is consistent with the Nagpal results. We\nhave included a brief discussion of these timescales in the manuscript.\n7) The results for the apparent interfacial tension are displayed\nin Figure 5H in the form of a histogram. This histogram exhibits\nat least two, maybe even three peaks. The authors should explain\nwhether or not they can interpret this histogram in terms of the\nunderlying molecular processes.\nDoes this histogram reflect, for instance, the competition between\ncovalent intra- and interchain contacts of the polymer chains?\nResponse:\nThank you for this comment. To address this question, we fully reworked this part\nof the manuscript and included new experimental results and developed new\nmodels that enable us to explain the observed heterogeneities. Figure 5H (now\nmoved to Figure S10 B) is a histogram of F/4πR*, which is the apparent interfacial\ntension for a liquid-like droplet (i.e., the PT40 samples). In +UV45 droplets,\ncrosslinking induces large viscoelasticity changes and thus deviation from the\ncapillary model. UV illumination is expected to alter surface tension too.\nTherefore, F/4πR* is a function of both viscoleasticity and surface tension\ntogether, and not surface tension alone. To demonstrate this directly, we\nmeasured surface tension for PT40 and +UV45 droplets and developed a modified\nversion of the capillary model, which includes viscoelastic effects. Our analysis\nshows that UV treatment increases interfacial tension, and the spread of F/4πR*\nis due to heterogeneity of surface tension to a large extent, with some contribution\nfrom heterogeneity of the viscoelastic moduli. We further elaborate on this point\nin Note S3, where we also include a propagation analysis of these effects. Finally,\nwe asked how the competition between covalent intra- and inter-chain contacts\naffects heterogeneity. To address this, we performed fusion experiments on\nPremix droplets and incorporated the results (along with the results of +UV45\ndroplets) into new theoretical models to estimate how the balance of inter- and\nintra-chain bonds influences noise propagation. Please see the last paragraph of\nthe Results section.\n8) Two typos on page 15: in line 399, Figure 5H should be replaced\nby Figure 5G, and in line 402, Figure 5I should be replaced by\nFigure 5H.\nResponse:\nThank you for this comment. We fixed these issues in the revised paper.\n9) Figure S1 is quite useful in order to understand the formation\nof the biomolecular condensates studied here, Therefore, the\nauthors might want to move Figure S1 into the main text.\nResponse:\nThank you for this comment. Following your advice, we included this figure in the\nmain text, as new Figure 1A.\nReviewer #4 (Remarks to the Author):\nIn a nutshell, the paper reports that UV illumination can alter the fusion times of\ncondensate droplets. I haven’t studied the literature exhaustively, but this does\nseem to me like a new and interesting discovery. I have comments:\nResponse:\nThank you very much for the critical assessment of the paper and positive remarks\non the findings. We carefully studied the comments and did our best to address\nthem all. In what follows, we describe the revisions we did to the manuscript:\n• I am not an expert in this specific area, but the authors state with great\nconfidence that the observed changes in condensate mechanics are due to\nthymine dimerization. I do not see direct evidence for this claim; at present it\nseems more like a plausible hypothesis rather than a demonstrated molecular\nmechanism.\nResponse:\nThank you for this comment. To address this comment, we decided to take three\nexperimental approaches to probe TT dimerization:\n1- We developed an assay in which we used anti-TT dimer antibodies to directly\nprobe TT dimer formation within the condensate phase (See Figure 1I).\n2- We performed UV spectroscopy, which revealed the spectroscopic signature\nof TT dimer formation (See Figure 1H).\n3- We developed a microscale thermophoretic (MST) assay, which reveals a\ndramatic change in DNA mobility upon UV treatment, which is expected for\ncross-linked DNA molecules (See Figure 4I).\nThese three lines of evidence prove the formation of the TT dimer in our study. The new\nresults are included in the revised version.\n• In Fig. 1G and H the authors analyze “neck length.” While this is a reasonable\nempirical observation, is there a quantitative framework that could connect this\nparameter to rheological or other mechanical quantities?\nResponse:\nThank you for this comment. In classical Newtonian fluids, neck growth during\nearly-stage coalescence scales with the ratio of surface tension to viscosity\nas in the viscous-dominated regime, where is the time since\n𝛾 𝜂\ncontact; this dynamics is not strongly influenced by elasticity [See P. J. Dekker et\n𝑟(𝑡) ∝ (𝛾/𝜂)𝑡 𝑡\nal., Phys. Rev. Lett. 128, 028004 (2022)], and so looking at the neck growth in time\nwith a higher spatial and temporal resolution may provide information about the\nviscosity. However, highly time- and space-resolved analysis is challenging in\ncondensate assays, and the dense viscoelastic environment of the condensates\ncomplicates interpretation.\nIn our study, the previously named “neck length” corresponds to a 2D projection\nof the contact region, which relates to the 3D neck radius in a complex way (and\ndiffers from the early-stage neck radius used in scaling relations). To avoid\nconfusion, we have renamed this parameter as “chord length” in the revised\nmanuscript. We focus on relative comparisons of chord length under different\nconditions rather than extracting absolute rheological parameters from it.\nExtended UV illumination increases chord length, reflecting slower relaxation due\nto higher viscoelasticity, which also allows doublets with larger chord lengths to\nbe more readily captured during imaging. Rheological properties are instead\nquantified directly via SPM measurements. We have clarified these points in the\nrevised manuscript to contextualize chord length as an empirical, indirect\nindicator of mechanical state.\n• I find the introduction poorly written and the results written with a somewhat\noverstated style. For example:\no Micropipette aspiration does not, in general, require the use of tracer particles.\nResponse:\nThank you for this comment. We agree that this part of the intro was not fully clear\nand in some parts overstated. We extensively revised the introduction and the\ndiscussion sections to improve clarity and tone.\nRegarding micropipette aspiration, you are correct that it does not require tracer\nparticles. The original statement was an error introduced during writing and does\nnot reflect our understanding of the technique. Our intention was to highlight its\nlimitations in force range and experimental throughput, and the revised\nmanuscript now states this accurately.\no To my knowledge, thymine dimerization involves the formation of covalent\nbonds, which necessarily alters the chemical composition. Therefore, the\nsentence claiming that chemical composition remains unchanged should be\nrevised.\nResponse:\nThanks for this comment. We agree that addition or rearrangement of chemical\nbonds is indeed a chemical change. Our intention in using the phrase “preserving\nchemical composition” was to distinguish our approach from more common\nstrategies in which condensates are chemically modified through the\nintroduction of new molecules or crosslinkers, which alter elemental\ncomposition. In our study, no additional molecules or crosslinkers are introduced\ninto the condensate, and salt concentrations remain unchanged. Thus, while\ncovalent connectivity is modified, the elemental (atomic) composition of the\ncondensates remains unchanged. We have revised the text to clarify this\ndistinction while still conveying our intended point more accurately.\nRevised introductory phrase reads:\n“…. while preserving chemical composition —that is, without adding new\nmolecules and without changing atomic composition.”\nRevised phrase in the discussion reads:\n“… light can be leveraged to program the connectivity and internal organization\nof a nucleic acid-based condensate system, thereby changing condensate\nmechanics with no alterations to atomic (elemental) composition.”\no The text contains multiple “first-of-its-kind” statements that are unnecessary.\nResponse: Thank you for this comment. We fixed this issue in the revised version and\nremoved all such references.\no Prior analogous work should be acknowledged, for instance the use of AFM to\nmeasure the material properties of objects as large as condensates\n(https://www.nature.com/articles/nphys4104).\nResponse: Thank you for this comment. To address this issue, we added the suggested\nreference and a few others, including a recent technical paper that covered prior work\nextensively.\no The paper is about material properties and their relation to molecular\nconformations, but does not cite the significant very recent progress in this area\nfrom the Banerjee, Mittal, and Schuler groups.\nResponse: Thank you for this comment and for bringing this interesting study to\nour attention. We agree that significant recent progress by the Banerjee, Mittal,\nand Schuler groups is relevant to our study. We have now included citations to\nthese works in the revised manuscript.\n• Line 332 seems to be referring to Figure 5B. Please check.\nResponse: Thank you for this comment. We addressed it in the revised version.\n• Finally, I find it puzzling that no condensates are reported in the “0h” condition.\nCould it be that phase separation does occur, but the droplets are arrested at\nsizes below the diffraction limit of the imaging system?\nResponse: We thank the reviewer for this insightful comment. Indeed, we cannot\ncompletely rule out the presence of condensates smaller than the diffraction limit during\nthe early phases of the “0 h” condition. Capturing the very first moments under UV\nillumination is practically challenging, as small objects typically remain floating and\nrequire time to settle onto the substrate before they can be imaged.\nTo address this, we took two approaches (See Figure S4):\n1) Real-time phase contrast imaging of objects on the substrate, during UV\nillumination, from time = 0 to time = 45 min.\n2) Microfluidic Imaging (MFI) analysis, which allows measurement of particle size\nand circularity while particles are freely floating. This was performed after the UV\nillumination was completed.\nOur phase contrast imaging revealed the formation of tiny condensate-like droplets at\nearly stages, which then quickly transitioned to aggregate-like structures within the time\nframe of the experiment. MFI analysis revealed particles with heterogeneous sizes and\ncircularities. A subset of these particles appeared somewhat circular, but notably less\ncircular than all condensates observed in our study under other conditions.\nFinally, we note that TT dimer formation in the dilute phase occurs on a very short\ntimescale (See our note in the Results section), which implies that condensation\nformation may be affected almost immediately upon UV exposure. As such, while the\ncondensate-to-aggregate transition is a process we observe, direct nucleation and\ngrowth of the aggregate states may also occur. However, capturing and characterizing\nsmall and transient structures is technically challenging. We have revised the\nmanuscript to acknowledge this possibility explicitly and to highlight the limitations\nimposed by the diffraction limit of optical microscopy, and incorporated our new\nexperimental data."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have largely addressed our concerns in the revised manuscript. We only have the following suggestions:\n1. Figure 1 in the response letter show a ~5 fold difference between two coating conditions. The data do not exclude the\npossibility the coating can have a significant effect the measured viscoelasticity of condensates. We think the authors should\nbe more cautious about this point and include this result as a supporting information in the manuscript.\n2. We were unable to locate the power density values of the UV illumination.\n3. The authors claim that SPM has higher range of forces compare to OT and micropipette aspiration without reporting the\nforce range of SPM. But this is not supported by any direct evidence.\n4. The number of samples and independent measurements are missing in several figures, including figure 2 and s2.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have responded to all concerns raised in my previous report and made appropriate changes in the revised\nmanuscript. The manuscript is now suitable for publication in Nature Communications.", "reviewer_4": "Good for publication."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have largely addressed our concerns in the revised manuscript.\nResponse: We thank the referee for the constructive feedback on our manuscript and\nare glad that the revisions were found satisfactory.\nWe only have the following suggestions:\n1. Figure 1 in the response letter show a ~5 fold difference between two coating\nconditions. The data do not exclude the possibility the coating can have a significant\neffect the measured viscoelasticity of condensates. We think the authors should be\nmore cautious about this point and include this result as a supporting information in the\nmanuscript.\nResponse: Thanks. We agree that surface properties does influence mechanical\nproperties as seen in our data. As suggested by the referee, we included the rheology\ndata on PF127-coated surface (presented in our previous response letter) as a new\npanel to Figure S2.\n2. We were unable to locate the power density values of the UV illumination.\nResponse: The power density is given in the second paragraph of Results section. It\nreads as follows:\nFor UV treatment, samples were prepared over a total period of 4 hours and placed under a\nUVC lamp (UVP UVG-11, l=254 nm; 1120µW/cm² at the sample) (Figure 1D), with UV\nillumination applied during the final 30 (+UV30) or 45 (+UV45) minutes49.\n3. The authors claim that SPM has higher range of forces compare to OT and\nmicropipette aspiration without reporting the force range of SPM. But this is not\nsupported by any direct evidence.\nResponse: Thanks. We included the force range available through SPM in the Methods\nsection.\n4. The number of samples and independent measurements are missing in several\nfigures, including figure 2 and s2.\nResponse: We thank the reviewer for this comment. We have thoroughly reviewed the\nmanuscript and incorporated the relevant sample size information where appropriate.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank the referee for the constructive feedback on our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have responded to all concerns raised in my previous report and made\nappropriate changes in the revised manuscript. The manuscript is now suitable for\npublication in Nature Communications.\nResponse: We thank the referee for the constructive feedback on our manuscript. We\nare pleased that the referee is satisfied with our revisions and has recommended\npublication of the work.\nReviewer #4 (Remarks to the Author):\nGood for publication.\nResponse: We thank the referee for the constructive feedback on our manuscript. We\nare pleased that the referee is satisfied with our revisions and has recommended\npublication of the work."}]

Decision letter