Characterization of LLPS Using Single-Molecule Fluorescence Imaging

Microscopy is an effective tool for visualizing the structure and composition of biomolecular condensates. Based on the cutting-edge single-molecule fluorescence microscopy platform, CD BioSciences offers customized single-molecule fluorescence experimental procedures to probe protein-RNA interactions under liquid-liquid phase separation (LLPS).

Many biomolecular condensates, including nucleoli and stress granules, are formed through routine multivalent interactions between dynamic multivalent proteins and proteins, proteins and RNA, or RNA and RNA. These molecular interactions allow LLPS nucleation and determine the properties of the condensates, such as size and mobility. Various variants of fluorescence microscopy are now available to label target structures. The development of fluorescence microscopy methods has opened up new areas for the study of intracellular biomolecular condensates, as fluorescence provides higher contrast than bright-field microscopy. For example, fluorescent photobleaching recovery (FRAP) is widely used to quantify the dynamic properties of membraneless organelles.

Customized Services

Our single-molecule fluorescence imaging platform is widely applicable to the imaging of biomolecular condensates. We provide various fluorescence techniques to tag target proteins, such as fluorescent dyes, immunolabeling, fluorescent fusion proteins, self-labeling tags, enzyme labeling, fluorescent unnatural amino acids, etc. In addition, we can label several target molecules with different fluorescent dyes and analyze the interactions of several proteins or even biomolecular condensates in the same experiment.

Attractively, CD BioSciences offers the following three complementary single-molecule methods to help customers analyze interactions on the basis of biomolecular condensates. All three methods are performed using total internal reflection fluorescence (TIRF) microscopy, and each method utilizes different principles to provide unique and complementary information to probe protein-RNA interactions at single-molecule resolution.

• Single-Molecule Fluorescence Resonance Energy Transfer (smFRET)
  This method is suitable for monitoring protein-induced RNA conformation changes. We use donor and acceptor dyes (e.g. Cyanine3 and Cyanine5) to double-label RNA and monitor protein-independent changes in RNA conformation by measuring FRET.
• Protein-Induced Fluorescence Enhancement (smPIFE)
  This method is suitable for measuring protein-RNA encounters. We use individual fluorophores 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.
• Nucleation Experiments
  We provide nucleation experiments to label proteins to monitor protein binding and accumulation on RNA by detecting a gradual increase in fluorescence signal, as well as a gradual decrease in signal when fluorophores are randomly photobleached. In addition, we optimize this approach to enable differential labeling of two proteins to test the co-localization of different proteins on the same RNA.

Experimental Procedure for Single-Molecule Fluorescence Imaging Characterization of LLPS

(1) Preparation of single-molecule slides

• Single molecules of RNA are tethered to PEG (polyethylene glycol) passivated surfaces by fluorescence.
• RNA concentration (50 -100 pM) was used to achieve the required density for single-molecule detection.
• The RNA substrate is designed as an 18-base-pair partial duplex for tethering to the surface, and the single-stranded RNA sequence of interest is positioned in solution away from the surface.
• Protein influx into single-molecule carriers.

(2) Probe different aspects of protein-RNA interactions according to the labeling scheme.

(3) Interpret the resulting data.

Advantages of the Single-Molecule Fluorescence Imaging Platform

• A powerful tool for analyzing protein-RNA interactions.
• Can provide structural and kinetic information in real time.
• No time or population averaging is required.
• Can characterize properties such as size, shape and mobility of biomolecular condensates.

CD BioSciences offers comprehensive single-molecule methods for studying RNA-protein condensates. Our team of experts has developed detailed protocols for preparing single-molecule slides, performing the experiments described above, and interpreting the resulting data. We will ensure that the labeling scheme does not affect the structure or function of the biomolecular condensates. If you are interested in our services, please do not hesitate to contact us for more information.