Lipid vesicles chaperone an encapsulated RNA aptamer

Abstract

The organization of molecules into cells is believed to have been critical for the emergence of living systems. Early protocells likely consisted of RNA functioning inside vesicles made of simple lipids. However, little is known about how encapsulation would affect the activity and folding of RNA. Here we find that confinement of the malachite green RNA aptamer inside fatty acid vesicles increases binding affinity and locally stabilizes the bound conformation of the RNA. The vesicle effectively 'chaperones' the aptamer, consistent with an excluded volume mechanism due to confinement. Protocellular organization thereby leads to a direct benefit for the RNA. Coupled with previously described mechanisms by which encapsulated RNA aids membrane growth, this effect illustrates how the membrane and RNA might cooperate for mutual benefit. Encapsulation could thus increase RNA fitness and the likelihood that functional sequences would emerge during the origin of life.

Fig. 1

Schematic representation of the MG aptamer in different conditions. a Exposed to ‘empty’ vesicles and b encapsulated inside vesicles. Comparisons between these two conditions isolate the effect of confinement vs. chemical interaction with the membrane

Fig. 2

MG aptamer affinity in fatty acid vesicles. a Encapsulation in MA vesicles (blue triangles) results in increased affinity compared to aptamer exposed to empty vesicles (gray squares). Representative binding curves are shown. Curve fits to the Hill equation are shown as lines (blue: K_D = 2.5 ± 0.2 μM; gray: K_D = 6.8 ± 1.4 μM). Encapsulation in b MA:MAOH (blue triangles: K_D = 2.1 ± 0.2 μM; gray square: K_D = 5.2 ± 0.1 μM) or c MA:GMM (blue triangles: K_D = 2.4 ± 0.1 μM; gray square: K_D = 5.1 ± 0.1 μM) vesicles shows a similar effect. Values given are mean ± standard deviation

Fig. 3

MG aptamer affinity in phospholipid vesicles. Encapsulation also increases the binding affinity of MG aptamer to MG in the phospholipid vesicles of DOPG (blue triangles: K_D = 7.1± 0.2 μM; gray square: K_D = 20 ± 1.7 μM) (a) or POPC (blue triangles: K_D = 18 ± 1.4 μM; gray square: K_D = 48 ± 8.6 μM) (b). Values given are mean ± standard deviation

Fig. 4

Melting transitions of the MG aptamer. a Fluorescence response of the MG aptamer with temperature in the presence of saturating MG concentrations (∼3-fold above the K_D), for aptamer exposed to empty MA vesicles (gray squares) and aptamer inside MA vesicles (blue triangles). Shown are representative data along with the curve fit to the Boltzmann sigmoidal equation. Note that because the fluorescence of the aptamer exposed to empty vesicles does not show saturation in the measured temperature range, the point of half-maximal measured intensity does not necessarily correspond to the estimated T_t. b CD response (at 264 nm) of the dye-bound MG aptamer at varying temperatures in buffer without vesicles (T_m = 71 ± 2 °C). Values given are mean ± standard deviation

Fig. 5

Conformational probing of the MG aptamer. a Representative SHAPE gel (MG aptamer exposed to empty MA vesicles). The first eight lanes show SHAPE reactions containing NMIA at increasing MG concentrations. The (−) lane shows a negative control containing 20 μM MG but in which no NMIA was added. The two rightmost lanes show A and U sequencing reactions performed using ddTTP and ddATP as chain terminators, respectively. The sequencing lanes give bands that are one nucleotide longer than the corresponding NMIA lanes. b Scheme showing the aptamer SHAPE construct and the MG-aptamer structure determined previously. The red cross in the center indicates the position of the MG ligand. Base pairing in stem regions (solid black lines) and other interactions (dotted lines) are shown. c Leftward shift in band intensity ratio between A30 to A31 at varying concentrations of MG when the aptamer is encapsulated in MA vesicles (blue triangles) vs. exposed to empty MA vesicles (gray squares). Representative concentration series are shown. Lines show curve fits to the Boltzmann equation

Fig. 6

MG aptamer affinity in the presence of different solutes. a The crowded conditions of 18% dextran (blue triangle, K_D = 1.8 ± 0.4 μM) lowers the K_D compared to 18% glucose (gray square, K_D = 3.6 ± 0.4 μM). b The transition temperature (T_t) of the MG aptamer (SHAPE construct) also increases in the presence 18% dextran (T_t = 28 ± 1.1 °C, blue triangles) compared to 18% glucose (T_t = 18 ± 0.8 °C, gray squares). c Binding curve of the MG aptamer in DOPG vesicles without Mg²⁺ in the medium (gray square: outside vesicle, K_D = 20 ± 1 μM; blue triangle: inside vesicle, K_D = 9.3 ± 2.4 μM). Values given are mean ± standard deviation