COMPLEX COACERVATES AS EXPERIMENTAL MODEL SYSTEMS FOR PREBIOTIC COMPARTMENTS AND CELLULAR BIOCONDENSATES
Restricted (Penn State Only)
- Author:
- Choi, Saehyun
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 15, 2022
- Committee Members:
- Paul Cremer, Major Field Member
Christine Keating, Chair & Dissertation Advisor
Philip Bevilacqua, Major Field Member
Philip Bevilacqua, Program Head/Chair
Christopher House, Outside Unit & Field Member - Keywords:
- Coacervates
liquid-liquid phase separation
origin of life
protocell
biocondensates
RNA chemistry
thermodynamics - Abstract:
- Cells compartmentalize biomolecules to fascilitate a spatiotemporal control of biological processes by providing distinctive microenvironments. Membraneless organelles, one type of compartment in cells, have been suggested to play pivotal roles in RNA localization and controlling biological processes by transient formation via liquid-liquid phase separation. As such, coacervates, membraneless compartments formed by liquid-liquid phase separation, have been of particular interest as model prebiotic compartments due to their physicochemical similarity to cellular membraneless organelles and their remarkable ability to encapsulate other molecules. Coacervates composed of polymers, proteins and RNAs with long lengths and stoichiometric charge ratios are most commonly studied since they tend to produce more stable coacervates owing to greater multivalency. On early Earth, however, more complex mixtures of various sized, low molecular weight molecules may have been more prevalent across a wide range of geological conditions. Therefore, it remains unclear how those factors would influence coacervates as compartments for their further biological processes. In this dissertation, I discuss my works to investigate coacervate stability under prebiotic-relevant environments and propose several important properties that may allow them to serve as biological compartments such as providing microenvironments, efficient encapsulation and altered thermodynamics of RNA chemistry, and an intrinsic buffering capacity. Chapter 1 discusses the physical chemistry of membraneless compartments and their roles that can relate to prebiotic compartments in more detail. By quantitative and thermodynamic analysis, I demonstrated that coacervates composed of oligopeptides and nucleotides are simple yet sufficient to maintain primitive functions as compartments under conditions from spring water to ocean vents by modulating interaction modes of coacervate molecules. Chapter 2 explores how prebiotic-relevant short oligopeptides can undergo phase separation to form coacervates providing different local apparent pHs and levels of RNA partitioning and structures inside. Coacervates composed of shorter polyions have better encapsulation of RNAs while maintaining RNA secondary structures, suggesting that the relative lengths of coacervate molecules to RNAs play an important role in their ability to act as RNA compartments. Chapter 3 continues to investigate the mechanism of the local apparent pH of coacervates by considering the effects of asymmetric lengths of polyions and various salt concentrations with polyelectrolyte phase separation theory, based on the transfer matrix method. Theoretical modeling confirms our hypothesis that the apparent pH difference between coacervate and dilute phase (outside of coacervates, supernatant phase) originated from the local interactions of pH indicators with polycations and can be modulated by lengths of polyion pairs. With knowledge gained from Chapter 2 and 3, we expanded these oligopeptide-based coacervates towards further functionalities as compartments of multiphase droplets (Chapter 4) or by introducing changes in solution conditions that mimic prebiotically relevant geological conditions (Chapter 5 and Chapter 6). Chapter 4 illustrates how oligopeptide-based multiphase complex coacervates can contribute to altered thermodynamics of RNA chemistry in each phase, that may provide an explanation for the underlying physical chemistry of RNA in multiphase biocondensates in cells. By comparing the thermodynamics of RNAs of multiphase to single-phase coacervates that have similar peptide contents in coacervate phase, I showed that multiphase complex coacervates can provide additional RNA duplex destabilization owing to coupled thermodynamics of RNA partitioning and RNA duplex destabilization in each phase of multiphase droplets. Chapter 5 illustrates the ability of complex coacervates to resist the changes in concentrations of phase separating molecules and RNAs under a wide range of pH and salt conditions, suggesting its primitive role as molecular buffering compartments. Required salt conditions for buffering coacervate molecules and RNAs were opposite, and these counterintuitive results were explained by modulated interaction modes between coacervate molecules upon condition changes. Chapter 6 explores the possibility of the coexistence of complex coacervates under simulated hydrothermal vent conditions that have pH gradients and FeS minerals. Lastly, Chapter 7 discusses general conclusions and future directions based on the works presented in this thesis. The works presented in this thesis reveal the primitive functions of coacervates as biological compartments. Coacervates can provide distinctive microenvironments, accumulate RNAs with phase-specific RNA chemistry, and buffer against environmental changes. Such aspects of coacervates within prebiotic conditions are important to assess geological boundary conditions that could favor the emergence of life. These investigations could provide the foundation for the underlying principles of the functions of coacervates as prebiotic compartments that could convey the rudimentary physical chemistry of biocondensates in extant cells.