Analysis of Protein-RNA Interactions Underlying Liquid-Liquid Phase Separation

The cellular localization, function and physical properties of biomolecular cohesions depend on multivalent interactions between the constituent molecules. Proteomics and transcriptomics studies have shown that cohesions are usually highly enriched in RNA and RNA-binding proteins, highlighting the importance of protein-RNA interactions. These molecular interactions underlie the liquid-liquid phase separation (LLPS) that occurs in biomolecular condensates and determine the properties of cohesions, such as size and mobility. A good understanding of protein-RNA interactions is available to provide supporting information for the characterization and spatial organization of condensates.

Fig. 1. RNA identity drives condensate properties. (Ganser L R, et al., 2023)

Customized Services

Our scientists use powerful single-molecule fluorescence tools to probe the protein-RNA interactions behind LLPS, providing you with structural and kinetic information in real time, without the need for time or population averaging. Based on our advanced total internal reflection fluorescence (TIRF) microscopy platform, CD BioSciences offers the following three complementary single-molecule methods to analyze protein-RNA interactions underlying liquid-liquid phase separation, helping customers understand the initial stages of cohesion formation, encompassing properties such as size, shape, and mobility. These three methods utilize different principles to provide unique and complementary information to probe protein-RNA interactions with single-molecule resolution.

• Single-Molecule Fluorescence Resonance Energy Transfer (smFRET)
  We use donor and acceptor dyes to dual-label RNA and monitor protein-independent changes in RNA conformation by measuring FRET.
• Protein-Induced Fluorescence Enhancement (PIFE)
  We use a single fluorophore to label RNA and monitor the fluorescence intensity to track protein binding. The position of the fluorophore on the RNA can be changed to determine the initial contact site.
• Single-Molecule Nucleation Experiments (SMNE)
  We monitor protein binding and accumulation on nucleating RNA by labeling proteins with a gradual increase in fluorescence signal, as well as a gradual decrease in signal when the fluorophore is randomly photobleached.

Since the interaction between non-coding RNA (ncRNA) and phase-separated structures is essentially transient, it is difficult to capture by conventional methods. We provide in situ single-molecule high-resolution localization and counting methods to accurately measure the interactions between phase-separated RNA and proteins in living cells.

Process of Single-Molecule Fluorescence Methods to Analyze Protein-RNA Interactions

(1) Prepare single-molecule slides. The RNA molecules are tethered to the single-molecule surface via biotin-neutravidin linkage via PEG (polyethylene glycol) passivation. The RNA concentration (50-100 pM) is used to achieve the density required for single-molecule detection. The RNA substrate is designed as an 18-base-pair partial duplex for tethering to the surface. Single-stranded RNA sequences of interest were localized in solution away from the surface.

(2) Analysis of protein-RNA interactions. Proteins are flowed into single molecule carriers and different aspects of protein-RNA interactions are probed according to the labeling scheme.

(3) Interpret the resulting data.

We develop customized experimental procedures for single-molecule fluorescence experiments to probe the protein-RNA interactions behind LLPS. Three complementary experimental approaches can elucidate the molecular view of protein-RNA interactions that drive the formation of ribonucleoprotein condensation from multiple perspectives. If you are interested in our services, please do not hesitate to contact us for more information.