Diverse Evolutionary Power of Nucleic Acid Libraries Carrying Different Information Content

在化学部生命过程化学(CLP)计划的支持下，应用分子进化基金会的elisa Biondi博士将研究可能作为复制大分子的前体的化学系统，如在地球和其他可能存在生命的地方发现的RNA和DNA。构成地球上当前生命的大分子只由所有可能的化学构件中的一小部分组成。Biondi博士的目标是提供关于只有一小部分分子如何进化并持久存在于当前大分子系统的见解。利用体外进化(在实验室进行的进化实验)的力量，Biondi博士的实验室将表征潜在的益生菌系统，以深入了解系统中的信息密度(构建块的数量)、信息量(显示最低活性所需的聚合物的最小长度)和信息多样性(构建块和/或化学基团的类型)如何影响潜在益生菌系统的性能和持久性。该项目将提供关于进化核酸文库所需信息密度的基本情报，并阐明许多基本问题，这些问题涉及在我们的星球或其他星球上发生的益生菌分子进化的限度和方式。此外，该项目的结果将为利用体外进化开发应用于生物医学、诊断、基因工程和生物修复等领域的分子工具的广大科学家提供宝贵的信息。Biondi博士的外展活动将主要集中在鼓励本科生，特别是女性和代表性不足的少数族裔，追求STEM职业生涯。此外，Biondi博士将参加以科学领域女性问题为中心的焦点小组，并将继续与3至5年级的学生进行虚拟会面，向他们介绍天体生物学和普通科学。该项目将调查哪些类型的核酸化学系统更适合向催化功能进化，并可能作为当代催化剂的前体。这项工作将使用生物化学工具和高通量测序数据来比较不同信息密度的核酸文库的实验室体外进化。核酸库的化学多样性将通过Biondi博士的实验室开发的人工扩展遗传信息系统(Aegis)进行调整。首先，将进行三个平行的体外选择能够切割RNA底物的RNA裂解DNAzyme。起始文库将包括一个25N的随机区域和由标准DNA或宙斯盾DNA组成的引物结合位点(PBS)，两侧是标准DNA。第三个选择将使用具有标准25N区域的文库，但在PBS中使用Aegis，这些文库已在FfAME上展示，以改进自配对并产生更清洁的PCR。通过这种方法，通过消除该领域数十年来一直受到嘲讽的一些实验室偏见，预计这项工作不仅将改善选择实验的输出，还将获得对以前被技术偏见“埋葬”的基于序列的适应度进化的额外洞察。在下一阶段，将重复初始实验，起始文库带有19、30、40和最多45个随机位置。一旦对每个选择的生化和高通量测序结果进行比较，这些分析将提供另一种水平的信息：预计不仅具有不同信息含量的文库将以不同的方式进化和探索序列空间，而且序列选项的长度差异也将提供不同的进化路线。此外，将比较在随机区、引物结合部位或分子的5‘和3’端内携带不同类型修饰的文库进行的选择的输出，目的是获得哪些基团和哪些分子组合对特定功能具有最佳适合性的综合看法。评选比较的指标将包括实现功能所需的周期数、催化参数(Kobs、Km、Kcat、周转率)以及序列适合度和富集值。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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 fordecades, 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.