Binding and disruption of phospholipid bilayers by supramolecular RNA complexes

Abstract

In an RNA world, RNAs would have regulated traffic through normally impermeable bilayer membranes. Using selection-amplification we previously found RNAs that bind stably and increase the ionic conductance of phospholipid membranes at high Mg 2؉ and Ca 2؉ concentrations. Now selection in reduced divalents yields RNAs that bind phosphatidylcholine liposomes under conditions closer to physiological. Such affinity for phospholipid membranes requires interactions between RNAs. In fact, we detected no functional monomeric membrane-binding RNAs. A membraneactive end-to-end heterotrimer consisting of 2 RNA 9 and 1 RNA 10 is defined by nucleotide protection, oligonucleotide competition, and mutant analysis. Oligomers of the heterotrimer bind stably, cause release of liposome-encapsulated solutes, and disrupt model black membranes. Individual RNA molecules do not show any of these activities. This novel mechanism of RNA binding to lipid membranes may not only regulate membrane permeability, but suggests that arrays of catalytic or structural RNAs on membranes are plausible. Finally, a selection met only by RNA complexes evokes new possibilities for selection-amplification itself.T he RNA world hypothesis (1) suggests that ancestral RNAs played roles presently taken by both nucleic acids and proteins. To show that RNA might serve essential membrane functions, we used selection-amplification to isolate RNA molecules with affinity for phospholipid liposomes (2). Some RNAs bound stably and increased the ionic permeability of liposomes, as well as the plasma membranes of cultured human cells. Analysis of individual isolated RNAs suggested that specific RNA sequences and folds are needed for membrane activity. In fact, one RNA appeared to bind in only one of two prevalent conformers. Earlier reports detected weaker, nonspecific interactions between membranes and nucleic acids (3), but these selected RNAs were the first stably bound nucleic acids with measurable membrane effects.The goal of the present study was to elucidate the mechanism of RNA binding to lipid membranes at moderate concentrations of divalent cations, closer to those found in cells and tissues. We have selected (4, 5) RNAs capable of efficiently binding to pure phosphatidyl choline liposomes, and we investigated RNA structure and interactions with liposomes and black membranes. Surprisingly, binding to the liposome surface requires formation of complexes between RNAs. Active multimer formation occurs via pairing of varied selected complementary sequences in weakly structured terminal regions, including ''kissing loops'' (6). Experimental ProceduresSelection. Incubation of internally 32 P-labeled RNA [1,000 pmol (cycles 1-4) and 100 pmol (cycles 5-11), 30 l] with liposomes (20 l, 20 mg͞ml) was performed in 50 mM Hepes (pH 7.0), 50 mM NaCl, 5 mM MgCl 2 , and 2 mM CaCl 2 at room temperature for 5 min, followed by gel filtration on a Sephacryl S-1000 (Amersham Pharmacia) 1-ml column. To decrease nonspecific sorption, the column was presaturated with liposomes (1...

Visualization of membrane RNAs

“…Following such an evolutionary rationale, RNAs that can bind, change the permeability, and disrupt phospholipid bilayers have been isolated using selection-amplification (Khvorova et al 1999;Vlassov et al 2001). The oligomerizing heterotrimer RNA (9:9:10) n ( Fig.…”

“…The oligomerizing heterotrimer RNA (9:9:10) n ( Fig. 1; Vlassov et al 2001) was one particular example, found to bind stably to the exposed face of a phosphatidylcholine vesicle. Because many RNA complexes with similar activities were isolated, with no sequences in common, many RNA structures exist that bind phospholipid membranes.…”

“…Below, we use fluorescence and atomic force microscopy (AFM) to characterize the molecular forms and disposition of the membrane-binding RNA 9:9:10 complex (see below; Vlassov et al 2001), both free and within a phospholipid membrane.…”

The selection of aptamers specific for membrane molecular targets

“…The selection of RNA aptamers was performed in low ionic-strength buffer (with a low concentration of monovalents, and a physiological concentration of divalents) against DOPC molecules forming 100-nm liposomes [53]. During the selection, the liposome-bound RNAs were separated from the unbound ones using gel chromatography.…”

Membrane self‐assembly processes: Steps toward the first cellular life

“…At first, the requirements imposed on early membranes by the lack of protein transport assemblies, and of stabilizing structures, such as cytosomal support proteins or cell walls suggests relatively permeable membrane boundaries which are best modeled by fattyacid vesicles (see Table 1). As the early cells began to synthesize polymeric molecules (perhaps RNA fragments (Khvorova et al, 1999;Vlassov et al, 2001), small polypeptides (Ghadiri et al, 1994;Oliver and Deamer, 1994;Clark et al, 1998;Kim et al, 1998), or other molecules that could mediate simple transport processes), more robust mixed amphiphile membranes that are modeled by short-chain phospholipid/fatty acids mixtures (such as DMPC/MA) would have replaced them. Finally, as populations of microbial organisms increasingly were able to produce their own specific amphiphiles, membranes of more homogenous composition would have appeared.…”