Selection of bifunctional RNAs with specificity for arginine and lipid membranes.

The molecular mechanisms of selective RNA loading into exosomes and other extracellular vesicles are not yet completely understood. In order to show that a pool of RNA sequences binds both the amino acid arginine and lipid membranes, we constructed a bifunctional RNA 10Arg aptamer specific for arginine and lipid vesicles. The preference of RNA 10Arg for lipid rafts was visualized and confirmed using FRET microscopy in neuroblastoma cells. The selection-amplification (SELEX) method using a doped (with the other three nucleotides) pool of RNA 10Arg sequences yielded several RNA 10Arg(D) sequences, and the affinities of these RNAs both to arginine and liposomes are improved in comparison to pre-doped RNA. Generation of these bispecific aptamers supports the hypothesis that an RNA molecule can bind both to RNA-binding proteins (RBPs) through arginine within the RBP-binding site and to membrane lipid rafts, thus facilitating RNA loading into exosomes and other extracellular vesicles.

Binding of RNA Aptamers to Membrane Lipid Rafts: Implications for Exosomal miRNAs Transfer from Cancer to Immune Cells.

Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP-RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane.

Exosome-associated polysialic acid modulates membrane potentials, membrane thermotropic properties, and raft-dependent interactions between vesicles.

In mammals, polysialic acid (polySia) attached to a small number of transmembrane protein carriers occurs on the surface of plasma membranes of neural, cancer, immune, and placental trophoblast cells. Here, our goal was to demonstrate the presence of polySia on exosomes and its effect on membrane properties. We isolated exosomes and found that polysialylated exosomes in fetal bovine serum originate mostly from placental trophoblasts, while in calf bovine serum, they originate from immune cells. Enzymatic removal of polySia chains from the exosomal surface makes the membrane surface potential more positive, transmembrane potential more negative, and reduces the activation energy for membrane anisotropy changes. We demonstrate for the first time that exosomes could interact through polySia-raft interactions. We suggest that polysialylation of exosomal membrane can have a thermo-protecting effect and can modulate exosome-plasma membrane interactions.

Polysialic acid chains exhibit enhanced affinity for ordered regions of membranes.

Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-dependent plasma membrane-bound) for liquid-ordered- and liquid-disordered regions of lipid vesicle and neuroblastoma cell membranes. Our studies indicate that polySia chains have a higher affinity for ordered regions of membranes as determined by the dissociation constant values for polySia-lipid bilayer complex, the fluorescence intensity of polySia bound to giant vesicles, the polySia-to-membrane FRET signal at the plasma membrane of live cells, and the decrease of the FRET signals after Endo-N treatment of the cells. These results suggest that polysialylation may be one of the determinants of protein association with liquid-ordered membrane lipid raft domains.

Membrane potential-dependent binding of polysialic acid to lipid monolayers and bilayers.

Polysialic acids are linear polysaccharides composed of sialic acid monomers. These polyanionic chains are usually membrane-bound, and are expressed on the surfaces of neural, tumor and neuroinvasive bacterial cells. We used toluidine blue spectroscopy, the Langmuir monolayer technique and fluorescence spectroscopy to study the effects of membrane surface potential and transmembrane potential on the binding of polysialic acids to lipid bilayers and monolayers. Polysialic acid free in solution was added to the bathing solution to assess the metachromatic shift in the absorption spectra of toluidine blue, the temperature dependence of the fluorescence anisotropy of DPH in liposomes, the limiting molecular area in lipid monolayers, and the fluorescence spectroscopy of oxonol V in liposomes. Our results show that both a positive surface potential and a positive transmembrane potential inside the vesicles can facilitate the binding of polysialic acid chains to model lipid membranes. These observations suggest that these membrane potentials can also affect the polysialic acid-mediated interaction between cells.