EMT: Cell Death by Boolean Calculations with Antibodies

我们的目标是在接下来的三年里展示人工创造的分子的混合物，我们将称之为治疗性分子自动机，可以在细胞表面执行布尔计算，具有治疗应用的结果(即细胞死亡)。具体地说，我们将探索分子自动机的一个特定亚型，由将在细胞表面组装的基于抗体的组件的混合物组成，根据细胞表面标志物(M1、M2和M3)的存在或不存在来执行布尔逻辑运算，并根据计算的输出触发细胞死亡。我们的自动机的主要组件将是：(1)基于抗体或其片段或适配子的识别区域，将针对特定的细胞表面标记；(2)用于在识别区域之间建立通信的寡核苷酸；以及(3)将在抗体交联时触发药物释放的药物输送组件。作为我们的三个目标，我们将在接下来的三年里测试在细胞上实施三种不同程序(或布尔逻辑运算)的混合物：1.{M1ANDM2凋亡}；三组分自动机，其行动将导致抗体的交叉连接和仅在显示用于靶向B细胞淋巴瘤系的两种细胞表面标记的细胞中凋亡显著增加。{M1ANDM2药物释放}；另一个三组分自动机，类似于上面的一个，除了它还会触发一个小的细胞毒分子的释放。{M3ANDNOTM1药物释放}，一个三组分自动机，它将考虑保护因素。智能价值分子自动机是分子网络，根据他们识别的输入的存在或不存在以及他们实施的算法来改变状态。在这个提议中，我们研究能够分析多个细胞表面或疾病标记的下一代分子自动机。就我们所知，构建与寡核苷酸结合的抗体的布尔自动机是一个全新的想法。广泛的意义生物医学意义：我们的自动机有可能在广泛的疾病过程中有用。所有基于有针对性地消除细胞亚群(例如癌症、自身免疫性疾病)的疗法都将受益于一种以上的细胞表面标记的能力，因为很少有细胞表面标记仅限于一种细胞类型。在教育方面，我们有一个非常有成效的项目，让当地的高中生加入我们的团队。这个项目的一个学生最近是描述分子加法器的出版物的第一作者，还有几个学生将成为我们正在准备的关于复杂自动机的手稿的合著者。在这个项目中，我们计划让一名暑期学生在第一年进行针对一个细胞表面标记的适体的分离。第二个学生将在这个项目的第二年在体外优化非门操作所需的结构转换过程。他们将接受面向学术的博士后研究员的培训。跨学科方法。我们注意到这个项目的跨学科性质，它结合了纳米技术、电气工程和计算机科学、核酸生物化学、细胞生物学、免疫学和有机化学的概念。

It is our goal over the next three years to demonstrate that mixtures of artificially created molecules, which we shall call therapeutic molecular automata, can perform Boolean calculation on cell surfaces with therapeutically applicable outcomes (i.e., cell death).Specifically, we will explore a specific subtype of molecular automata, consisting of mixtures of antibody-based components that will assemble on a cell surface, perform Boolean logic operations based on the presence or absence of cell-surface makers (M1, M2, and M3) and, depending on the output of calculation, trigger cell death. Main components of our automata will be: (1) recognition regions based on antibodies or their fragment or aptamers that will target characteristic cell-surface makers; (2) oligonucleotides used to establish communications between recognition regions; and (3) drug delivery components that will trigger release of drugs upon cross-linking of antibodies. As our three aims, we will test over the next three years mixtures implementing three different programs (or Boolean logic operations) on cells:1. {M1ANDM2 Apoptosis}; a three-components automaton, the action of which will result in crosslinking of antibodies and significant increase in apoptosis only in cells that display both cell-surface makers used to target B-cell lymphoma lines.2. {M1ANDM2 Drug Release}; another three-component automaton, similar to the one above, except it will also trigger the release of a small cytotoxic molecule.3. {M3ANDNOTM1 Drug Release}, a three component automaton, which will take into account a protective factor.Intellectual meritMolecular automata are networks of molecules that change states according to the presence or absence of inputs they recognize and an algorithm they implement. In this proposal we study the next generation of molecular automata capable of analyzing multiple cellsurface or disease markers. To the best of our knowledge, the proposal to construct Boolean automata with antibodies conjugated to oligonucleotides is a completely novel idea.Broader SignificanceBiomedical Significance: Our automata have the potential to be useful in a wide range of disease processes. All therapies that are based on a targeted elimination of a subpopulation of cells (e.g. cancer, autoimmune diseases) will benefit from the ability of to take into account more than one cell-surface marker, because very few cell surface markers are limited to only one cell type.Educational Significance. Education-wise, we have a very productive program to integrate local high schools students into our group. One student from this program has been recently a first author on a publication describing molecular full adder, and several more students will be coauthors on a manuscript that we are preparing on complex automata. In this project we plan to involve one summer student during first year in the isolation of aptamer against one cell-surface marker. The second student will optimize in vitro the structure switching process needed for a NOT gate operation during the second year of this program. They will be trained by an academically oriented postdoctoral fellow.Interdisciplinary approach. We note the interdisciplinary nature of this project that combines concepts from nanotechnology, electrical engineering and computer science, biochemistry of nucleic acids, cell biology, immunology and organic chemistry.