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.

Effect of UV Radiation on Fluorescent RNA Aptamers’ Functional and Templating Ability

“…Interestingly, the final fluorescence after long UV exposure times was notably high (25±3 %) for the Spinach2 aptamer compared to the MG aptamer. To determine whether the rate of activity loss depended on the folding of the RNA, we irradiated the MG aptamer at 80 °C, which is well above the melting point (see also Figure B, below) . No significant change in rate or final fluorescence was observed (1.0±0.2 h −1 and 4±1 %, respectively; Figure B), thus indicating that the folded and unfolded states of this aptamer are similarly susceptible to activity loss through UV damage.…”

“…No significant change in rate or final fluorescence was observed (1.0±0.2 h −1 and 4±1 %, respectively; Figure B), thus indicating that the folded and unfolded states of this aptamer are similarly susceptible to activity loss through UV damage. We also tested whether encapsulation of the MG aptamer inside phospholipid vesicles (composed of 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC)), which promotes aptamer folding, affected the rate of activity loss. Encapsulation did not significantly affect the rate of loss of fluorescence (Figure S1 C); however, exposure at lower UV fluence resulted in less loss of fluorescence, as expected (Figure S1 D).…”

“…where F is the fluorescence, A is a pre‐exponential factor, k f is the rate constant of fluorescence decrease and t is the time of exposure. Encapsulation of the MG aptamer in POPC vesicles was performed as described previously …”

“…Reverse transcription assay for UV damage : The RNA used for the reverse transcription assay was generated from a DNA template (aptamer region in bold text): 5′‐TAATA CGACT CACTA TAGGG CCTTC GGGCC AA GGA TCCCG ACTGG CGAGA GCCAG GTAAC GAATG GATCC TCGAT CCGGT TCGCC GGATC CAAAT CGGGC TTCGG TCCGG TTC‐3′). This sequence contains a T7 promoter, 5′ and 3′ linker regions, and a 3′ reverse transcription (RT) primer binding site, as previously described . The 5′ and 3′ flanking sequences of 14 and 43 nucleotides allow every base of the MG aptamer to be evaluated in a sequencing gel.…”

“…The ssDNA template was obtained from IDT and amplified through 30 cycles of PCR by using KAPA Taq ReadyMix PCR kit (Kapa Biosystems) in a Bio‐Rad C1000 thermal cycler (forward primer sequence: 5′‐TAATA CGACT CACTA TAGGG CCTTC GG‐3′; reverse primer sequence: 5′‐GAACC GGACC GAAGC CCG‐3′). Transcription was performed by using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB) as previously described . Reverse transcription reactions were conducted as follows: irradiated RNA (10 μL) concentrated after ethanol precipitation (or 1 μL of 143 μ m transcript for sequencing reactions), reverse primer (2 μL, 2 μ m ), and dNTP mix (1 μL, 10 μ m ) were mixed, and the mixture was incubated at 65 °C for 5 min, then annealed on ice for 5 min.…”