Improving Surface Passivation for Biomolecular Studies

Biomolecular Condensates are specialized areas within our cells that gather and concentrate various biological molecules, acting like tiny compartments. Unlike traditional cell compartments, these do not have a surrounding membrane. The formation of these areas often happens through a process called liquid-liquid phase separation, where certain large molecules cluster together.

Studying these condensates has led to important discoveries in the understanding of cellular functions. Scientists often use advanced techniques, including fluorescence microscopy, to observe and analyze these condensates. However, when these experiments are conducted, a significant challenge arises: the interaction between the condensates and the glass surfaces used for observation can change the properties of the condensates and lead to unwanted background that complicates results.

This is problematic because if the condensates interact with the surface too much, they can spread out and lose their distinct properties. On the other hand, completely preventing the condensates from sticking to the surface can also affect experiments, as some degree of movement is necessary for observation. Therefore, finding the right balance in surface treatment is essential for successful biochemical studies.

#Current Passivation Techniques

A commonly used technique to reduce these unwanted interactions involves covering the glass surfaces with substances like methoxy polyethylene glycol (mPEG) and Bovine Serum Albumin (BSA). While this method can be useful, it has notable limitations. In many cases, it does not sufficiently prevent the condensates from spreading, especially in systems where the molecules have high adhesive properties. Additionally, residual interactions can create background noise in sensitive experiments like single-molecule imaging.

Moreover, the standard mPEG/BSA treatment is time-consuming and has multiple steps, which can be difficult for labs that do not specialize in this area. Surface properties can also vary significantly between different types of condensates and experimental conditions, which means scientists often need to adjust their methods for each new experiment.

Given these challenges, there is a clear need for a more efficient and effective method to passivate surfaces in studies involving biomolecular condensates.

#Self-assembly as a Solution

Self-assembly refers to the natural process where molecules organize themselves into stable structures without the need for any external guidance. Recent advancements in using Surfactants-molecules that can reduce surface tension-have shown promise in preventing unwanted binding of biomolecules to surfaces.

In our studies, we tested several non-ionic surfactants for their ability to passivate glass surfaces. One surfactant, Pluronic F127 (PF127), stood out as the most effective option for stopping non-specific binding of both the phase-separated condensates and surrounding biomolecules. This method was robust across different pH levels and salt conditions, maintaining the physical structure of the condensates.

Moreover, by combining PF127 with the Biotin-NeutrAvidin system, we could immobilize the condensates in a controlled manner. This allowed us to use various imaging techniques sensitive to movement, providing us with clearer observations of the biomolecular dynamics.

#Experimenting with PF127 for Passivation

#Screening Surfactants

To assess the potential of PF127 and other surfactants for surface passivation in studying biomolecular condensates, we began by treating glass slides with a hydrophobic coating. The goal was to see how well different surfactants could prevent the binding of biomolecules.

From our trials, we discovered that surfactants like Brij L23 and Tween 20 also showed good abilities to passivate surfaces. However, PF127 consistently performed better, allowing condensates to form droplets that maintained their shapes without unwanted spreading.

#PF127 Passivation Efficiency

The PF127 passivation process we developed is simpler and quicker than traditional methods. The treatment can be completed in about three hours with less than one hour of active work time, in contrast to the more than 15 hours needed for mPEG/BSA treatments.

When comparing PF127 with standard passivation techniques across different types of condensate systems, we found that PF127 consistently resulted in lower adhesion and higher contact angles. This means that PF127-treated surfaces were better at preventing unintended interactions with the condensates, allowing for more accurate observations.

#Performance in Sensitive Studies

Even when examining single molecules around condensates, PF127 passivation proved highly effective. The excess molecules in the surrounding solution often bind to surfaces and create background noise that can interfere with sensitive imaging techniques like single-molecule tracking. We found that PF127 greatly reduced this unwanted binding, allowing for better-quality data collection.

#Robustness of PF127 Passivation

We proceeded to test the durability of PF127 treatment under various experimental conditions, such as different washing volumes, pH levels, and salt concentrations. The results revealed that surfaces treated with PF127 could withstand significant washing without losing their passivation capabilities. This resilience is crucial for experiments requiring multiple washes or varying environmental conditions.

#Assessment of Surface Integrity

To confirm the stability of PF127-treated surfaces, we observed how they performed after being subjected to rigorous washing. The contact angles indicated that PF127 remained effective at preventing unwanted binding even after extensive rinsing. The treated surfaces also did not exhibit fluorescence under common imaging conditions, which is a desirable trait for clarity in observations.

#Immobilizing Condensates with Biotin-NeutrAvidin

While PF127 passivation allowed for great freedom of movement in condensates, we found that this could be a limitation in certain experimental setups. To counteract this, we introduced a method to immobilize the condensates using biotin-labeled BSA as anchor points.

This approach added a low-density arrangement of specific anchor points on the surface, allowing the condensates to attach at defined spots while retaining a high degree of surface passivation. We found that this did not negatively affect the movement or interactions of the biomolecules, indicating that PF127 passivation can work alongside anchoring methods effectively.

#Analyzing Binding Site Distribution

Using single-molecule imaging, we began to explore how polySIM molecules interacted within the polySUMO/polySIM condensates. After bleaching specific areas, we monitored how quickly fluorescent polySIM molecules re-entered the condensates. We noticed that the recovery rate of fluorescent signals varied between the edges and the centers of the condensates.

The edges had faster recovery and movement than the center, suggesting different densities of available binding sites. This observation offers insights into how molecular dynamics and interactions might differ within various parts of the condensates, hinting at complex behaviors that could influence cellular functions.

#Implications for Future Research

The PF127 self-assembly method we developed is not only efficient but also cost-effective, easily adapted for various experimental setups. Its straightforward application could expand access to complex biochemical studies for many labs and researchers.

By preventing non-specific binding and enabling controlled interactions, this method opens the door for exploring various biomolecular properties and their interactions within condensates. Future developments could further enhance this technique, potentially leading to new materials or combinations that improve surface properties even more.

#Conclusion

In summary, the introduction of PF127 as a surface passivation method offers a robust solution for studying biomolecular condensates. It effectively minimizes unwanted interactions, preserves the physical properties of condensates, and is adaptable to various experimental conditions. This advancement holds great potential for researchers seeking to understand the intricate dynamics of biomolecular processes within the cell.

As scientists continue to unravel the complexities of these condensates, the ease and effectiveness of PF127 passivation will likely pave the way for new discoveries and enhancements in our understanding of cellular mechanisms.