Recent Articles

Annual Review of Biophysics 53, 109

The relationship between genotype and phenotype, or the fitness landscape, is the foundation of genetic engineering and evolution. However, mapping fitness landscapes poses a major technical challenge due to the amount of quantifiable data that is required. Catalytic RNA is a special topic in the study of fitness landscapes due to its relatively small sequence space combined with its importance in synthetic biology. The combination of in vitro selection and high-throughput sequencing has recently provided empirical maps of both complete and local RNA fitness landscapes, but the astronomical size of sequence space limits purely experimental investigations. Next steps are likely to involve data-driven interpolation and extrapolation over sequence space using various machine learning techniques. We discuss recent progress in understanding RNA fitness landscapes, particularly with respect to protocells and machine representations of RNA. The confluence of technical advances may significantly impact synthetic biology in the near future.

Acc. Chem. Res. 2024, 57, 2058

Creating life from nonliving matter poses a significant challenge in chemistry and biophysics. The early "RNA World" hypothesis, where RNA carried out both genetic and catalytic functions, offers inspiration for simpler systems than today's complex life forms. Minimal cells could consist of simple vesicles containing prebiotic RNA metabolism. The unique environment within these vesicles, with altered diffusion and molecular concentrations, impacts RNA behavior in ways that are distinct from chemical interactions alone.

Research has shown that encapsulating RNA inside vesicles enhances RNA folding and activity due to excluded volume effects, independent of chemical interactions. This increased stability translates into greater ribozyme activity and can even rescue mutant ribozymes. High-throughput sequencing revealed that encapsulation benefits more active ribozyme variants, aligning with Fisher’s Fundamental Theorem of Natural Selection, which suggests that increased fitness variance accelerates evolutionary adaptation.

In vitro evolution experiments demonstrated that encapsulated ribozymes evolved more quickly toward high-activity sequences. These findings suggest that simple encapsulation within vesicles could significantly impact the evolutionary trajectory of RNA, potentially providing insights into the transition from RNA encapsulation to living systems.

Protein aggregation is an important problem for human health and biotechnology, Methods to modulate protein aggregation are therefore essential. One suggested method to modulate protein aggregation is the use of nucleic acid aptamers, that is, single-stranded nucleic acids that have been selected to specifically bind to a target. Previous studies in some systems have demonstrated that aptamers may inhibit protein aggregation, including for α-synuclein, a protein implicated in synucleinopathies. However, the mechanisms by which aptamers might affect or modulate aggregation have not been fully determined. In this study, we investigated the effect of an aptamer that binds α-synuclein oligomer, T-SO508, on α-synuclein aggregation in vitro using thioflavin T to monitor aggregation kinetics, and we performed atomic force microscopy, transmission electron microscopy, and analytical ultracentrifugation to characterize intermediate structures. The results indicated that T-SO508, but not control DNA sequences, extends the lag phase of aggregation and stabilizes formation of a small non-fibrillar aggregate complex. Attempts to use the aptamer-induced complexes to seed fibril formation did not in fact accelerate aggregation, indicating that these structures are off-pathway for aggregation. This study highlights a potential mechanism by which aptamers may modulate the aggregation properties of proteins.

UV damage to functional RNAs has been relatively little studied. We irradiated two fluorescent RNA aptamers and monitored the loss of activity, folding, and the kinetics of lesion accumulation. UV susceptibility was highly heterogeneous across the RNA sites; this indicates the importance of UV exposure as a selective pressure during the origin of life.

The organization of molecules into cells is believed to have been critical for the emergence of living systems. Early protocells likely consisted of RNA functioning inside vesicles made of simple lipids. However, little is known about how encapsulation would affect the activity and folding of RNA. Here we find that confinement of the malachite green RNA aptamer inside fatty acid vesicles increases binding affinity and locally stabilizes the bound conformation of the RNA. The vesicle effectively ‘chaperones’ the aptamer, consistent with an excluded volume mechanism due to confinement. Protocellular organization thereby leads to a direct benefit for the RNA. Coupled with previously described mechanisms by which encapsulated RNA aids membrane growth, this effect illustrates how the membrane and RNA might cooperate for mutual benefit. Encapsulation could thus increase RNA fitness and the likelihood that functional sequences would emerge during the origin of life.

To study the origin of life, synthetic biologists construct simple ‘protocells’, but previous models were not able to reproduce both genome and membrane sustainably. A recent advance feeds the protocells by vesicle fusion, suggesting a practical pathway for indefinite self-reproduction.

The environment of protocells might have been crowded with small molecules and functional and non-specific polymers. In addition to altering conformational equilibria, affecting reaction rates and changing the structure and activity of water, crowding might have enhanced the capabilities of protocells for evolutionary innovation through the creation of extended neutral networks in the fitness landscape.