Smart nucleic acid condensates for bottom-up synthetic biology
Author(s)
Fabrini, Giacomo
Type
Thesis
Abstract
Bottom-up synthetic biology aims at rationally engineering life-like behaviours, such as gene expression, energy production and communication, in synthetic entities assembled from bio-active components, termed artificial cells. The complex functions of biological cells often emerge from their heterogeneous sub-compartmentalised architecture, from the outer lipid membrane to inner organelles. Therefore, the capability to engineer programmable micro-compartments is key to developing biomimetic artificial cells.
While lipid, polymer and protein-based approaches have been proposed to replicate the semipermeable cellular membrane, solutions relying on coacervates, hydrogels and biomolecular condensates are gaining traction as versatile and tunable mimics of cellular membrane-less organelles. Nucleic acid nanotechnology has emerged as a prime route to develop biomimetic artificial cellular platforms thanks to the control it enables over structure and dynamics at the nano- and microscale. In particular, branched nucleic acid nanostructures (“nanostars”) have been extensively researched and exploited to build programmable nanomaterials, including liquid-like condensates, hydrogels and crystals, with potential applications in bottom-up synthetic biology.
In this thesis I investigate two approaches to engineer smart artificial cellular compartments based on nucleic acid condensates. The first strategy modularly combines the self-assembly robustness of amphiphilic DNA nanostars with the cation-responsiveness of non-canonical DNA G-quadruplexes. The resulting nanostructures can isothermally self-assemble into amphiphilic hydrogels upon addition of physiologically-relevant concentrations of potassium, and disassemble upon its chelation. The second approach exploits biological machinery to produce RNA nanostars from the transcription of DNA templates. The RNA nanostructures fold co-transcriptionally and self-assemble into condensates, both in bulk and in simple synthetic cells. The resulting RNA organelles can selectively recruit small molecules and proteins, and boast programmable number, morphology and mixing/demixing behaviours. Overall, my findings contribute to demonstrating the largely untapped potential that nucleic acid-based synthetic condensates hold as a modular platform for artificial cell engineering, potentially paving the way to advanced biomimetic functionalities.
While lipid, polymer and protein-based approaches have been proposed to replicate the semipermeable cellular membrane, solutions relying on coacervates, hydrogels and biomolecular condensates are gaining traction as versatile and tunable mimics of cellular membrane-less organelles. Nucleic acid nanotechnology has emerged as a prime route to develop biomimetic artificial cellular platforms thanks to the control it enables over structure and dynamics at the nano- and microscale. In particular, branched nucleic acid nanostructures (“nanostars”) have been extensively researched and exploited to build programmable nanomaterials, including liquid-like condensates, hydrogels and crystals, with potential applications in bottom-up synthetic biology.
In this thesis I investigate two approaches to engineer smart artificial cellular compartments based on nucleic acid condensates. The first strategy modularly combines the self-assembly robustness of amphiphilic DNA nanostars with the cation-responsiveness of non-canonical DNA G-quadruplexes. The resulting nanostructures can isothermally self-assemble into amphiphilic hydrogels upon addition of physiologically-relevant concentrations of potassium, and disassemble upon its chelation. The second approach exploits biological machinery to produce RNA nanostars from the transcription of DNA templates. The RNA nanostructures fold co-transcriptionally and self-assemble into condensates, both in bulk and in simple synthetic cells. The resulting RNA organelles can selectively recruit small molecules and proteins, and boast programmable number, morphology and mixing/demixing behaviours. Overall, my findings contribute to demonstrating the largely untapped potential that nucleic acid-based synthetic condensates hold as a modular platform for artificial cell engineering, potentially paving the way to advanced biomimetic functionalities.
Version
Open Access
Date Issued
2023-08-13
Date Awarded
01/11/2023
License URL
Advisor
Di Michele, Lorenzo
Sponsor
Department of Chemistry
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)