Incorporation of Basic α-Hydroxy Acid Residues into Primitive Polyester Microdroplets for RNA Segregation

Abstract

Nucleic acid segregation and compartmentalization were likely essential functions that primitive compartment systems resolved during evolution. Recently, polyester microdroplets generated from dehydration synthesis of various α-hydroxy acids (αHA) were suggested as potential primitive compartments. Some of these droplets can differentially segregate and compartmentalize organic dyes, proteins, and nucleic acids. However, the previously studied polyester microdroplets included limited αHA chemical diversity, which may not reflect the chemical diversity available in the primitive Earth environment. Here, we increased the chemical diversity of polyester microdroplet systems by combinatorially adding an αHA monomer with a basic side chain, 4-amino-2-hydroxybutyric acid (4a2h), which was incorporated with different ratios of other αHAs containing uncharged side chains to form combinatorial heteropolyesters via dehydration synthesis. Incorporation of 4a2h in the polymers resulted in the assembly of some polyester microdroplets able to segregate fluorescent RNA or potentially acquire intrinsic fluorescent character, suggesting that minor modifications of polyester composition can significantly impact the functional properties of primitive compartments. This study suggests one process by which primitive chemical systems can increase diversity of compartment "phenotype" through simple modifications in their chemical composition.

A material‐based panspermia hypothesis: The potential of polymer gels and membraneless droplets

“…[121][122][123] Membraneless droplets generated by liquid-liquid phase separation (LLPS), [114,124] such as coacervates [125] or aqueous two-phase systems, [126] have also shown the ability to segregate primitive biomolecules such as RNA or peptides. [127][128][129][130] While such systems can be cyclically assembled and disassembled (e.g., through modulation of environmental conditions such as pH, salt, or temperature [131][132][133] ), depending on the composition, membraneless droplets may have been more "leaky" than vesicles to encapsulated components. [116] Other primitive compartments, such as mineral pores, are very stable on long timescales and have been shown to promote polymerization of primitive genetic materials such as RNA, [134,135] but their structure is governed by its geochemical composition, and thus it is difficult to envision any dynamic structural changes on the short term.…”

“…Polymeric gels in particular could have potentially formed compartments similar to the membraneless droplets described above on prebiotic Earth, potentially through phase separation (or more appropriately, LLPS), which is a process in which liquid mixtures spontaneously separate into two liquid phases, [136] forming membraneless droplets. For example, polyester gels generated from dehydration synthesis (by heating) of alpha-hydroxy acid monomers have recently been shown to form membraneless microdroplets upon hydration in aqueous solution [127,128,137] (Figure 2). Other primitive polymers formed through dehydration polymerization have also shown similar behavior in their ability to form membraneless droplets.…”

“…While again, such a demonstration is based on non-gelmaterial-based droplets, previous demonstrations showed the ability for primitive gel-based droplets to acquire differential charges based on residue incorporation. [127,128]…”

Liquid Crystal Phase Assembly in Peptide-DNA Coacervates as a Mechanism for Primitive Emergence of Structural Complexity

“…Liquid-liquid phase separation (LLPS) is a ubiquitous process in modern cells that contribute to a variety of essential biological processes (Yoshizawa et al, 2020). Recently, LLPS such as polyester microdroplets (Chandru et al, 2020;Jia et al, 2021;Jia et al, 2019), aqueous two-phase systems (Mizuuchi & Ichihashi, 2020), or complex coacervates (Fares et al, 2020) have been proposed as primitive membraneless compartments at the origin of life. Complex coacervates in particular are produced from multivalent electrostatic binding of oppositely charged polymers, including those that may have been present on early Earth such as peptides or nucleic acids.…”

Increasing complexity of primitive compartments

“…Building such structures from the bottom up, such as through assembly of polymerization products generated from primitive wet-dry cycles [ 5 ], remains one mechanism to study the basic characteristics of such systems. Recently, polyesters generated from drying of simple prebiotic monomers can assemble to form membraneless droplets which can perform simple functions such as nucleic acid encapsulation [ 6 , 7 ]. As other “non-biological” polymers [ 8 ], along with polyesters [ 9 ], have shown the ability to form such membraneless compartments, focus should not be placed only on “biological” polymers such as nucleic acids and peptides from an origins of life perspective.…”