Template-directed RNA polymerization and enhanced ribozyme catalysis inside membraneless compartments formed by coacervates

Abstract

Membraneless compartments, such as complex coacervates, have been hypothesized as plausible prebiotic micro-compartments due to their ability to sequester RNA; however, their compatibility with essential RNA World chemistries is unclear. We show that such compartments can enhance key prebiotically-relevant RNA chemistries. We demonstrate that template-directed RNA polymerization is sensitive to polycation identity, with polydiallyldimethylammonium chloride (PDAC) outperforming poly(allylamine), poly(lysine), and poly(arginine) in polycation/RNA coacervates. Differences in RNA diffusion rates between PDAC/RNA and oligoarginine/RNA coacervates imply distinct biophysical environments. Template-directed RNA polymerization is relatively insensitive to Mg2+ concentration when performed in PDAC/RNA coacervates as compared to buffer, even enabling partial rescue of the reaction in the absence of magnesium. Finally, we show enhanced activities of multiple nucleic acid enzymes including two ribozymes and a deoxyribozyme, underscoring the generality of this approach, in which functional nucleic acids like aptamers and ribozymes, and in some cases key cosolutes localize within the coacervate microenvironments.

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

“…Herein, we describe the formation and photoswitchable behavior of light-responsive coacervate droplets prepared from mixtures of double-stranded DNAa nd an azobenzene cation. [5][6][7] These membrane-free molecularly crowded compartments seques-ter functional biomolecules [5,8] and support enzymatic activity, [9,10] protein folding, [11] RNAc atalysis, [12,13] and cell-free protein expression. Sequestration and release of captured oligonucleotides followt he dynamics of phase separation such that light-activated transfer,m ixing,h ybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets.O ur results open perspectives for the spatiotemporal control of DNAc oacervates and provide as tep towards the dynamic regulation of synthetic protocells.…”

“…[1,2] They play acrucial role for example in the compaction of polynucleotides and regulation of genetic expression. [5][6][7] These membrane-free molecularly crowded compartments seques-ter functional biomolecules [5,8] and support enzymatic activity, [9,10] protein folding, [11] RNAc atalysis, [12,13] and cell-free protein expression. [5][6][7] These membrane-free molecularly crowded compartments seques-ter functional biomolecules [5,8] and support enzymatic activity, [9,10] protein folding, [11] RNAc atalysis, [12,13] and cell-free protein expression.…”

Lipid Sponge Droplets as Programmable Synthetic Organelles

“…Nonetheless, the product yield is enhanced greatly by programming the droplets to sequester cathepsin K and by concentrating and co-localizing enzyme and substrate. Previously enhanced reaction rates have been observed by concentrating biomolecules in complex coacervates (4,39,40). We believe that the lipid sponge droplets will offer a general programmable scaffold for studying the effects of colocalization and confinement on enzymatic reactions.…”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…[66] Other factors including the ionic strength, [67] the order of addition of the coacervate components versus protein [68] or the polyelectrolyte ratio [67] weref urthers hown to affect protein sequestration. [75] Significantly,b yr econciling membrane-free compartmentalisation and RNA catalysis, these studies provide possible routes towardst he emergence of protocells capable of RNA-based catalytic activity and genetici nformation storage on the primordial Earth, in line with the RNA world hypothesis. [18,70] Chemical modifications have recently been used to increaset he propensityo fp roteins to undergo LLPS through, for example, protein supercharging [71] or covalent attachment of ionic polypeptidetags.…”