Utilizing nucleic strand displacement reactions for sensing and computation in living cells

该项目的总体目标是利用所谓的“链置换过程”(即动态替换RNA结构中的RNA链)在活细胞，特别是哺乳动物细胞中执行简单的计算。这将允许通过特定RNA分子的存在来读出细胞状态，并将能够通过改变转录水平或甚至通过修改细胞基因组来对其做出反应。我们的项目将建立在我们之前为CRISPR相关核酸酶Cas12a的链置换引导RNA(SD GRNAs)所做的工作的基础上，这些工作已经被证明在体外和细菌细胞内起作用。在活细胞的复杂环境中建立更强大的RNA“电路”带来了相当大的挑战，我们在这个项目中旨在解决其中的一些挑战。我们首先旨在建立哺乳动物细胞中高效链置换电路的设计规则。为了在哺乳动物细胞中有效地执行这样的过程，我们将探索各种RNA设计参数，这些参数预计会影响杂交和链置换动力学。我们进一步的目标是开发一组便于多输入电路的正交化元件，并最终使用设计规则来生成级联电路。我们还旨在了解对链置换过程动力学的其他影响，这些影响可能在细胞的复杂环境中发挥作用。为此，我们将在无细胞的情况下对细胞环境的几个方面进行实验建模。特别是，我们将研究大量竞争的DNA或RNA物种对链置换动力学的影响，但也会研究其他方面，如大分子拥挤。然后，我们的目标是扩展现有的基于核酸的反应电路的建模框架，以解释这些影响，并潜在地预测体内链置换过程的动力学。最后，我们希望将我们的见解应用于使用天然RNA作为哺乳动物细胞中链置换电路的输入。为了利用细胞RNA物种，我们将开发能够有效检测具有特定序列限制的长RNA分子的策略，其中将包括评估可用于杂交的序列结构域，以及与RNA结合蛋白的竞争。此外，我们将研究RNA分子在细胞内的定位和浓度对我们电路功能的影响。

The overall goal of the project is the utilization of so-called “strand displacement processes” (i.e., dynamic replacement of RNA strands in an RNA structure) to perform simple computations in living cells, and especially mammalian cells. This will allow the readout of the cellular state via the presence of specific RNA molecules and will enable responding to it by changing transcription levels or even by modification of the genome of the cell. Our project will build upon our previous work on strand-displacing guide RNAs (SD gRNAs) for the CRISPR associated nuclease Cas12a, which were already shown to work in vitro and inside bacterial cells. Building up more powerful RNA “circuits” in the complex environment of living cells poses considerable challenges, some of which we aim to address in this project.We first aim at establishing design rules for efficient strand displacement circuits in mammalian cells. In order to perform such processes in mammalian cells efficiently, we will explore various RNA design parameters that are expected to influence hybridization and strand displacement kinetics. We further aim at the development of a set of orthogonal components, which facilitate multi-input circuits, and finally use the design rules to generate cascaded circuits. We also aim at understanding additional influences on the kinetics of strand displacement processes that might play a role in the complex environment of a cell. To this end we will experimentally model several aspects of the cellular environment in a cell-free context. In particular, we will study the influence of large pools of competing DNA or RNA species on strand displacement kinetics, but also other aspects such as macromolecular crowding. We then aim to extend existing modeling frameworks for nucleic acid-based reaction circuits to account for these effects and potentially predict the kinetics of strand displacement processes in vivo. Finally, we wish to apply our insights to use natural RNA as input for strand displacement circuits in mammalian cells. In order to utilize cellular RNA species, we will develop strategies that allow efficient sensing of long RNA molecules with their specific sequence constraints, which will include an evaluation of sequence domains available for hybridization and also competition with RNA-binding proteins. Further, we will study the impact of intracellular localization and concentration of the RNA molecules on the functionality of our circuits.