在化学和生物物理学中,从非生命物质中创建生命系统是一个巨大的挑战。生命的早期历史可以从核酶建立的益生元“RNA世界”的想法中提供灵感,其中所有的遗传和催化活性都是由RNA执行的。这样一个系统可能比今天描述生活的相互依存的中心法则简单得多。同时,合作系统需要一种机制,如细胞区隔,以便生存和进化。因此,最小的细胞可能由包围益生元RNA代谢的简单囊泡组成。囊泡的内部体积由于其封闭的边界而成为独特的环境,这改变了大分子的扩散和可用体积,并改变了有效的分子浓度,在其他考虑中。这些物理效应在机械上不同于化学相互作用,如静电排斥,这也可能发生在膜边界和封装内容物之间。膜和RNA之间的间接和直接相互作用都会产生非直觉,模型原型细胞系统中的“紧急”行为。我们一直在研究膜囊泡内的包封将如何影响包封的RNA的折叠和活性。使用生物物理技术,如FRET,我们表征了囊泡内核酶的折叠和活性。模型原始细胞内的封装通常促进RNA折叠,与排除的体积效应一致,独立于化学相互作用。在所研究的两个不同系统(发夹状核酶和自氨基酰化RNA)中,这种能量稳定转化为增加的核酶活性。一个特别有趣的发现是封装可以拯救突变核酶的活性,这表明封装不仅会影响折叠和活性,还会影响进化。为了进一步研究这一点,我们开发了一种高通量测序试验来平行测量数千种核酶变体的氨基酰化动力学.结果揭示了与较差的变体相比,封装改善较好的核酶变体的趋势出乎意料。在进化过程中,这种效果会创造一个倾斜的运动场,可以这么说,这将为已经高活动量的变种带来额外的健身收益。根据费雪的自然选择基本定理,适应度变化的增加应该表现为更快的进化适应。这一预测在体外进化过程中得到了实验证实,我们观察到,当最初不同的核酶群体被封装在囊泡中时,它们更快地收敛到最活跃的序列。本帐户中的研究扩大了我们对新兴原始细胞行为的理解,通过展示如何简单地将RNA截留在囊泡中,这可能在囊泡形成过程中自发发生,可能会深刻影响RNA的进化格局。由于复制和选择的指数动力学,即使是活动和功能的微小变化也可能导致重大的进化后果。通过仔细研究最小但令人惊讶的复杂原始细胞的细节,我们可能有一天会追踪到一条从封装的RNA到生命系统的通路。
ConspectusCreating a living system from nonliving matter is a great challenge in chemistry and biophysics. The early history of life can provide inspiration from the idea of the prebiotic \"RNA World\" established by ribozymes, in which all genetic and catalytic activities were executed by RNA. Such a system could be much simpler than the interdependent central dogma characterizing life today. At the same time, cooperative systems require a mechanism such as cellular compartmentalization in order to survive and evolve. Minimal cells might therefore consist of simple vesicles enclosing a prebiotic RNA metabolism.The internal volume of a vesicle is a distinctive environment due to its closed boundary, which alters diffusion and available volume for macromolecules and changes effective molecular concentrations, among other considerations. These physical effects are mechanistically distinct from chemical interactions, such as electrostatic repulsion, that might also occur between the membrane boundary and encapsulated contents. Both indirect and direct interactions between the membrane and RNA can give rise to nonintuitive, \"emergent\" behaviors in the model protocell system. We have been examining how encapsulation inside membrane vesicles would affect the folding and activity of entrapped RNA.Using biophysical techniques such as FRET, we characterized ribozyme folding and activity inside vesicles. Encapsulation inside model protocells generally promoted RNA folding, consistent with an excluded volume effect, independently of chemical interactions. This energetic stabilization translated into increased ribozyme activity in two different systems that were studied (hairpin ribozyme and self-aminoacylating RNAs). A particularly intriguing finding was that encapsulation could rescue the activity of mutant ribozymes, suggesting that encapsulation could affect not only folding and activity but also evolution. To study this further, we developed a high-throughput sequencing assay to measure the aminoacylation kinetics of many thousands of ribozyme variants in parallel. The results revealed an unexpected tendency for encapsulation to improve the better ribozyme variants more than worse variants. During evolution, this effect would create a tilted playing field, so to speak, that would give additional fitness gains to already-high-activity variants. According to Fisher\'s Fundamental Theorem of Natural Selection, the increased variance in fitness should manifest as faster evolutionary adaptation. This prediction was borne out experimentally during in vitro evolution, where we observed that the initially diverse ribozyme population converged more quickly to the most active sequences when they were encapsulated inside vesicles.The studies in this Account have expanded our understanding of emergent protocell behavior, by showing how simply entrapping an RNA inside a vesicle, which could occur spontaneously during vesicle formation, might profoundly affect the evolutionary landscape of the RNA. Because of the exponential dynamics of replication and selection, even small changes to activity and function could lead to major evolutionary consequences. By closely studying the details of minimal yet surprisingly complex protocells, we might one day trace a pathway from encapsulated RNA to a living system.