Diverse Evolutionary Power of Nucleic Acid Libraries Carrying Different Information Content

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Dr. Elisa Biondi of Foundation for Applied Molecular Evolution, Inc. will study chemical systems that could have served as precursors to the replicating macromolecules, such as RNA and DNA, found on Earth and other places where life could exist. The macromolecules that make up current life on Earth are composed of only a small subset of all possible chemical building blocks.

Dr Biondi aims to provide insights into how only a small subset of molecules evolved and persisted into the current macromolecular systems. Using the power of in-vitro evolution (evolutionary experiments performed in the laboratory), Dr. Biondi’s laboratory will characterize potential prebiotic systems to provide insights into how the information density in the system (the number of building blocks), information amount (the minimum length of the polymer needed to display minimal activity), and information diversity (the types of building blocks and/or chemical groups) affect the performance and persistence of potential prebiotic systems.

This project will provide fundamental intelligence on the information density requirement for an evolving nucleic acid library and shed light on many fundamental questions regarding the limits and ways in which prebiotic molecular evolution took place on our planet or others. Additionally, results from this project will provide precious information to the large community of scientists who make use of in-vitro evolution to develop molecular tools applied in fields such as biomedicine, diagnostics, genetic engineering, and bioremediation.

Dr. Biondi’s outreaching activities will focus mainly on encouraging undergraduates, especially women and underrepresented minorities, to pursue STEM careers. Moreover, Dr.

Biondi will participate in focus groups centered on the issue of women in science and will continue her virtual encounters with 3rd to 5th grade students to introduce them to astrobiology and general sciences.

This project will investigate what types of nucleic acid chemical systems are more suited for evolution towards catalytic functions and might have served as precursors of contemporary catalysts. The work will compare laboratory in-vitro evolution of nucleic acid libraries with different information density using biochemistry tools and high-throughput sequencing data.

The chemical diversity of nucleic acid libraries will be adjusted with the Artificially Expanded Genetic Information System (AEGIS), developed in Dr. Biondi’s laboratory. First, three parallel in-vitro selections of RNA-cleaving DNAzymes able to cut an RNA substrate will be performed.

The starting libraries will include a 25N random region and primer binding sites (pbs) composed of either standard or AEGIS DNA, flanked by standard DNA. A third selection will use libraries with a standard 25N region, but AEGIS in the pbs, which have been shown at FfAME to improve self-pairing and yield cleaner PCRs. With this method, by removing some laboratory biases that have taunted the field for

decades, it is expected that the work will not only improve the output of selection experiments, but also to gain extra insights into sequence-based fitness evolution previously “buried” under techniques biases. In the next phase, initial experiments will be repeated with starting libraries carrying 19, 30, 40, and up to 45 random positions. These analyses will provide another level of information once biochemical and high throughput sequencing results for each selection will be compared: it is expected that not only will libraries with different information content evolve and explore the sequence space differently, but that a length difference in sequence options will also provide different evolutionary routes.

Further, outputs of selections performed with libraries carrying different types of modifications inside the random region, primer binding sites, or 5’ and 3’ ends of the molecules will be compared, with the goal of obtaining a comprehensive view of which groups and which combination of molecules have the best fitness towards a specific function. Metrics for selection comparison will include number of cycles required to achieve function, catalysis parameters (kobs, kM, kcat, turnover rates), and sequence fitness and enrichment values.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.