Compartmentalised RNA catalysis in membrane-free coacervate protocells

Abstract

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via complex coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides.

Fig. 1

Cleavage of the FRET substrate under different conditions. a HH-min (black) and the FRET substrate (red). b Gel electrophoresis of RNA cleavage in bulk coacervate phase (CM-Dex : PLys, 4:1 final molar ratio); 0.5 μM of FRET substrate was incubated with 1 μM of (i) HH-min, (ii) HH-mut or (iii) no ribozyme in bulk phase (25 °C, 60 min). Samples were analysed by denaturing PAGE followed by fluorescence imaging. The lack of in-gel quenching of the FRET substrate likely results from modifications of BHQ1 during PAGE. c Real-time cleavage kinetics in 10 mM Tris-HCl pH 8.3 and 4 mM MgCl₂. (i) A monoexponential fit (Methods, Eq. 3) (grey line) to kinetic data (grey dots) and residuals of the fit (inset); (ii) mean of the individual fits to each experiment (Blue line) with the standard deviation of the mean of the fits (grey data points) (N = 5). d Cleavage in bulk coacervate phase (normalised to the amount of cleaved product at t = 530 min from gel electrophoresis). (i) Biexponential fit (Methods, Eq. 4) (dark grey line) to experimental data (grey dots) with the residuals (inset); (ii) mean biexponential fit (orange) of individual fits (N ≥ 5). Grey data points represent the standard deviation (N = 5) from the experimental data

Fig. 2

FRAP of bulk coacervate phase. Bulk coacervate phase (CM-Dex:PLys (4:1 final molar ratio) containing either (i) 0.36 μM FAM-substrate or (ii) 0.36 μM TAM-HH-min. a Output frames from confocal imaging (63×) are shown at t = −0.5 s before bleaching, directly after bleaching (magenta circle, t = 0 s) and t = 13 s after bleaching. The fluorescence intensity was normalised against a reference (green circle) and fit to standard equations. Scale bars are 5 μM. b Plots of normalised FRAP data for HH-min (ii) and FAM-substrate (ii) show the standard deviation (grey, N = 10) and fit (blue) from the same bleach spot radius. c Diffusion coefficients and viscosities obtained from b. Mean and standard deviations are from at least two different samples with analysis from ≥14 bleach spots for each experiment

Fig. 3

RNA catalysis in coacervate microdroplets. a (i) Wide-field optical microscopy images of CM-Dex:PLys (4:1 final molar ratio) coacervate microdroplets prepared in cleavage buffer (1 μM of HH-min and 0.5 μM FRET substrate). Fluorescence microscopy images at t = 0 min (ii) and t = 900 min (iii) show an increase in FAM fluorescence (see inset). Scale bars are 20 μM. b Background corrected and volume/endpoint normalised fluorescence intensity of droplets. Standard deviation of kinetics from 12 micro-droplets (grey) with the mean biexponential fit (blue) and residuals (inset)

Fig. 4

Localisation and retention of RNA within coacervate droplets. a Schematic of localisation experiments where CM-Dex:PLys (4:1 final molar ratio) coacervate droplets containing 0.36 μM (final concentration) TAM-HH-min were loaded into one end of a capillary channel (1). Droplets containing 0.36 μM FRET-substrate were loaded into the other end of the channel (3). b Wide-field optical microscopy images obtained using a 100 × oil immersion lens in (i) bright field and fluorescence mode using filters for (ii) TAM or (iii) FAM. Images were captured in regions 1, 2 and 3 at t = 500 min (scale bar: 20 μM). c FAM fluorescence intensity. Shaded regions represent the standard deviation of at least seven droplets