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.

The role of lipid rafts in vesicle formation.

The formation of membrane vesicles is a common feature in all eukaryotes. Lipid rafts are the best-studied example of membrane domains for both eukaryotes and prokaryotes, and their existence also is suggested in Archaea membranes. Lipid rafts are involved in the formation of transport vesicles, endocytic vesicles, exocytic vesicles, synaptic vesicles and extracellular vesicles, as well as enveloped viruses. Two mechanisms of how rafts are involved in vesicle formation have been proposed: first, that raft proteins and/or lipids located in lipid rafts associate with coat proteins that form a budding vesicle, and second, vesicle budding is triggered by enzymatic generation of cone-shaped ceramides and inverted cone-shaped lyso-phospholipids. In both cases, induction of curvature is also facilitated by the relaxation of tension in the raft domain. In this Review, we discuss the role of raft-derived vesicles in several intracellular trafficking pathways. We also highlight their role in different pathways of endocytosis, and in the formation of intraluminal vesicles (ILVs) through budding inwards from the multivesicular body (MVB) membrane, because rafts inside MVB membranes are likely to be involved in loading RNA into ILVs. Finally, we discuss the association of glycoproteins with rafts via the glycocalyx.

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs.

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.

Specific binding of VegT mRNA localization signal to membranes in Xenopus oocytes.

We have studied the interaction of a VegT mRNA localization signal sequence with the membranes of the mitochondrial cloud in Xenopus oocytes, and the binding of the VegT mRNA signal sequence to the lipid raft regions of the vesicles bounded by ordered and disordered phospholipid bilayers. RNA preference for the membranes of the mitochondrial cloud was confirmed using microscopy of a fluorescence resonance energy transfer from RNA molecules to membranes. Our studies show that VegT mRNA has a higher affinity for ordered regions of lipid bilayers. This conclusion is supported by the dissociation constant measurements for RNA-liposome complex and the visualization of the FRET signal between giant vesicles and RNA. Our data indicate that these affinities are sensitive and distinct to the location of the localization elements within the VegT mRNA localization signal structure. Therefore, specific binding of VegT mRNA localization signal sequence to membranes can be responsible for polarized distribution of VegT mRNA in Xenopus oocytes. We suggest that the mechanism of this binding can involve the interaction of the localization elements within the VegT mRNA signal sequence with lipid raft regions of the mitochondrial cloud membranes, thereby utilizing localization elements as novel lipid raft-binding RNA motifs.

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.

Cholera Toxin Subunit B for Sensitive and Rapid Determination of Exosomes by Gel Filtration.

We developed a sensitive fluorescence-based assay for determination of exosome concentration. In our assay, Cholera toxin subunit B (CTB) conjugated to a fluorescence probe and a gel filtration technique (size-exclusion chromatography) are used. Exosomal membranes are particularly enriched in raft-forming lipids (cholesterol, sphingolipids, and saturated phospholipids) and in GM1 ganglioside. CTB binds specifically and with high affinity to exosomal GM1 ganglioside residing in rafts only, and it has long been the probe of choice for membrane rafts. The CTB-gel filtration assay allows for detection of as little as 3 × 108 isolated exosomes/mL in a standard fluorometer, which has a sensitivity comparable to other methods using advanced instrumentation. The linear quantitation range for CTB-gel filtration assay extends over one order of magnitude in exosome concentration. Using 80 nM fluorescence-labeled CTB, we quantitated 3 × 108 to 6 × 109 exosomes/mL. The assay ranges exhibited linear fluorescence increases versus exosome concentration (r2 = 0.987). The assay was verified for exosomal liposomes. The assay is easy to use, rapid, and does not require any expensive or sophisticated instrumentation.

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.

Selection of Membrane RNA Aptamers to Amyloid Beta Peptide: Implications for Exosome-Based Antioxidant Strategies.

The distribution of amyloid beta peptide 42 (Aß42) between model exosomal membranes and a buffer solution was measured. The model membranes contained liquid-ordered regions or phosphatidylserine. Results demonstrated that up to ca. 20% of amyloid peptide, generated in the plasma (or intracellular) membrane as a result of proteolytic cleavage of amyloid precursor proteins by ß- and γ-secretases, can stay within the membrane milieu. The selection of RNA aptamers that bind to Aß42 incorporated into phosphatidylserine-containing liposomal membranes was performed using the selection-amplification (SELEX) method. After eight selection cycles, the pool of RNA aptamers was isolated and its binding to Aß42-containing membranes was demonstrated using the gel filtration method. Since membranes can act as a catalytic surface for Aß42 aggregation, these RNA aptamers may inhibit the formation of toxic amyloid aggregates that can permeabilize cellular membranes or disrupt membrane receptors. Strategies are proposed for using functional exosomes, loaded with RNA aptamers specific to membrane Aß42, to reduce the oxidative stress in Alzheimer's disease and Down's syndrome.

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.

Exosomes and other extracellular vesicles in neural cells and neurodegenerative diseases.

The function of human nervous system is critically dependent on proper interneuronal communication. Exosomes and other extracellular vesicles are emerging as a novel form of information exchange within the nervous system. Intraluminal vesicles within multivesicular bodies (MVBs) can be transported in neural cells anterogradely or retrogradely in order to be released into the extracellular space as exosomes. RNA loading into exosomes can be either via an interaction between RNA and the raft-like region of the MVB limiting membrane, or via an interaction between an RNA-binding protein-RNA complex with this raft-like region. Outflow of exosomes from neural cells and inflow of exosomes into neural cells presumably take place on a continuous basis. Exosomes can play both neuro-protective and neuro-toxic roles. In this review, we characterize the role of exosomes and microvesicles in normal nervous system function, and summarize evidence for defective signaling of these vesicles in disease pathogenesis of some neurodegenerative diseases.

Poly(U) RNA-templated synthesis of AppA.

Simple nucleotide templating activities are of interest as potential primordial reactions. Here we describe the acceleration of 5'-5' AppA synthesis by 3'-5' poly(U) under normal solution conditions. This reaction is apparently templated via complementary U:A base-pairing, despite the involvement of two different RNA backbones, because poly(U), unlike other polymers, significantly stimulates AppA synthesis. These interactions occur in moderate (K(+)) and (Mg(2+)) and are temperature sensitive, being more efficient at 10°C than at 4°C, but absent at 20°C. The reaction is only slightly pH sensitive, despite potentially relevant substrate pKa's. Kinetic data explicitly support production of AppA by interaction of stacked 2MeImpA and pA nucleotides paired with a single molecule of U template. At a lower rate, AppA can also be produced by a chemical reaction between 2MeImpA and pA, without participation of poly(U). Molecular modeling suggests that 5'-5' joining between stacked or concurrently paired A's can occur without major departures from normal U-A helical coordinates. So, coenzyme-like 5'-5' purine dinucleotides might be readily synthesized from 3'-5' RNAs with complementary sequences.

Mechanisms of RNA loading into exosomes.

Upon fusion of multivesicular bodies (MVBs) with the plasma membrane, intraluminal vesicles (ILVs) are released into the extracellular space as exosomes. Since the lipid composition of the exosomal membrane resembles that of raft microdomains, the inward budding process involves the raft-like region of the MVB limiting membrane. Although published research suggests that cellular RNAs may be selectively sorted into exosomes, the molecular mechanisms remain elusive. In this review, we suggest that there is a continuous interaction of cellular RNAs with the outer (cytoplasmic) surface of MVBs and that the selection for incorporation of these RNAs into ILVs is based on their affinity to the raft-like region in the outer layer of the MVB membrane.

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.

Human tRNA(Sec) associates with HeLa membranes, cell lipid liposomes, and synthetic lipid bilayers.

We have shown previously that simple RNA structures bind pure phospholipid liposomes. However, binding of bona fide cellular RNAs under physiological ionic conditions is shown here for the first time. Human tRNA(Sec) contains a hydrophobic anticodon-loop modification: N6-isopentenyladenosine (i6A) adjacent to its anticodon. Using a highly specific double-probe hybridization assay, we show mature human tRNA(Sec) specifically retained in HeLa intermediate-density membranes. Further, isolated human tRNA(Sec) rebinds to liposomes from isolated HeLa membrane lipids, to a much greater extent than an unmodified tRNA(Sec) transcript. To better define this affinity, experiments with pure lipids show that liposomes forming rafts or including positively charged sphingosine, or particularly both together, exhibit increased tRNA(Sec) binding. Thus tRNA(Sec) residence on membranes is determined by several factors, such as hydrophobic modification (likely isopentenylation of tRNA(Sec)), lipid structure (particularly lipid rafts), or sphingosine at a physiological concentration in rafted membranes. From prior work, RNA structure and ionic conditions also appear important. tRNA(Sec) dissociation from HeLa liposomes implies a mean membrane residence of 7.6 min at 24°C (t(1/2) = 5.3 min). Clearly RNA with a 5-carbon hydrophobic modification binds HeLa membranes, probably favoring raft domains containing specific lipids, for times sufficient to alter biological fates.

Membrane oligo- and polysialic acids.

Polysialic acid (polySia) and oligosialic acid (oligoSia) chains are linear polysaccharides composed of sialic acid monomers. The majority of biological poly/oligoSia chains are bound to membranes. There is a large diversity of membrane poly/oligoSia in terms of chain length, occurrence, biological function, and the mode of membrane attachment. Poly/oligoSia can be anchored to a membrane via a phospholipid (polySia in bacteria), a glycosphingolipid (oligoSia in gangliosides), an integral membrane glycoprotein, or a glycoprotein attached to a membrane via glycosylphosphatidylinositol. In eukaryotic cells, the attachment of a poly/oligoSia chain to the membrane anchor is usually through α-2,3-glycosidic linkage to a galactose. In prokaryotic cells this attachment is proposed to occur through glycosidic linkage to the phosphate group of a phospholipid. Both long polySia chains attached to membrane proteins and short oligoSia attached to glycosphingolipids or membrane proteins are frequently found in neural membranes. In humans, poly/oligoSia is involved in development and plasticity of the brain, pathophysiology of schizophrenic brains, cancer metastasis, neuroinvasive potential of pathogenic bacterial strains, and the immune response. Biological roles of poly/oligoSia are based on its ability to modulate repulsive and attractive interactions between two molecules, and its ability to modulate membrane surface charge density, pH at the membrane surface, and membrane potentials.

The effect of long-chain bases on polysialic acid-mediated membrane interactions.

Negatively-charged polysialic acid (polySia) chains are usually membrane-bound and are often expressed on the surface of neuroinvasive bacterial cells, neural cells, and tumor cells. PolySia can mediate both repulsive and attractive cis interactions between membrane components, and trans interactions between membranes. Positively-charged long-chain bases are widely present in cells, are often localized in membranes and can function as bioactive lipids. Here we use Langmuir monolayer technique, fluorescence spectroscopy and electron microscopy of lipid vesicles to study the role of a simple long-chain base, octadecylamine (ODA), in both cis and trans interactions mediated by polySia in model membranes composed of ODA and dioleoylphospatidycholine (DOPC). When added free to an aqueous solution, polySia increases the collapse pressure of ODA/DOPC monolayers, reduces the effect of ODA on the limiting molecular area, inverses the values of excess area per molecule and of excess free energy of mixing from positive to negative, and induces fusion of ODA/DOPC vesicles. These results suggest that a polySia chain can act as a multi-bridge that mediates cis interactions between different components of a lipid membrane, disrupts membrane aggregates, and mediates trans interactions between lipids in apposing membranes. These observations imply that polySia in cellular systems can act in a similar way.

Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes.

Noncovalent DIDS binding to Band 3 (AE1) protein in human erythrocyte membranes, modified by non-penetrating, water soluble 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC), was studied at 0°C in the presence of 165 mM KCl. Under experimental conditions applied up to (48 ± 5) % of irreversible chloride self-exchange inhibition was observed. The apparent dissociation constant, KD, for "DIDS-Band 3" complex, determined from the chloride transport experiments, was (34 ± 3) nM and (80 ± 12) nM for control and EAC-treated resealed ghosts, respectively. The inhibition constant, Ki, for DIDS was (35 ± 6) nM and (60 ± 8) nM in control and EAC-treated ghosts, respectively. The reduced affinity for DIDS reversible binding was not a result of negative cooperativity of DIDS binding sites of Band 3 oligomer since Hill's coefficients were indistinguishable from 1 (within the limit error) both for control and EAC-treated ghosts. By using tritium-labeled DIDS, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate ([(3)H]DIDS), the association rate constant, k(+1) (M(-1)s(-1)), was measured. The mean values of (4.3 ± 0.7) × 10(5) M(-1)s(-1) for control and (2.7 ± 0.7) × 10(5) M(-1)s(-1) for EAC-treated ghosts were obtained. The mean values for K(D), evaluated from [(3)H]DIDS binding measurements, were (37 ± 9) nM and (90 ± 21) nM for control and EAC-modified ghosts, respectively. The results demonstrate that EAC modification of AE1 reduces about 2-fold the affinity of AE1 for DIDS. It is suggested that half of the subunits are modified near the transport site by EAC.

The selection of aptamers specific for membrane molecular targets.

A growing number of RNA aptamers have been selected experimentally using the SELEX combinatorial approach, and these aptamers have several advantages over monoclonal protein antibodies or peptides with respect to their applications in medicine and nanobiotechnology. Relatively few successful selections have been reported for membrane molecular targets, in contrast to the situation with non-membrane molecular targets. This review compares the procedures and techniques used in selections against membrane proteins and membrane lipids. In the case of membrane proteins, the selections were performed against soluble protein fragments, detergent-membrane protein mixed micelles, whole cells, vesicles derived from cellular membranes, and enveloped viruses. Liposomes were used as an experimental system for the selection of aptamers against membrane lipids. RNA structure-dependent aptamer binding for rafts in lipid vesicles was reported. Based on the selected aptamers against DOPC and the amino acid tryptophan, a specific passive membrane transporter composed of RNA was constructed. The determination of the selectivity of aptamers appears to be a crucial step in a selection, but has rarely been fully investigated. The selections, which use whole cells or vesicles derived from membranes, can yield aptamers not only against proteins but also against membrane lipids.

Simple, recurring RNA binding sites for L-arginine.

Seven new arginine binding motifs have been selected from a heterogeneous RNA pool containing 17, 25, and 50mer randomized tracts, yielding 131 independently derived binding sites that are multiply isolated. The shortest 17mer random region is sufficient to build varied arginine binding sites using five different conserved motifs (motifs 1a, 1b, 1c, 2, and 4). Dissociation constants are in the fractional millimolar to millimolar range. Binding sites are amino acid side-chain specific and discriminate moderately between L- and D-stereoisomers of arginine, suggesting a molecular focus on side-chain guanidinium. An arginine coding triplet (codon/anticodon) is highly conserved within the largest family of Arg sites (72% of all sequences), as has also been found in minimal, most prevalent RNA binding sites for Ile, His, and Trp.

Polysialic acid can mediate membrane interactions by interacting with phospholipids.

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.