EMT: Cell Death by Boolean Calculations with Antibodies

我们在未来三年的目标是证明人工创造的分子的混合物，我们将称之为治疗分子自动机，可以在细胞表面上执行布尔计算，并获得治疗适用的结果（即，具体来说，我们将探索分子自动机的一个特定亚型，它由基于抗体的组分的混合物组成，这些组分将在细胞表面上组装，基于细胞表面标记（M1，M2和M3）的存在或不存在执行布尔逻辑运算，并根据计算的输出触发细胞死亡。我们的自动机的主要组成部分将是：（1）基于抗体或其片段或适体的识别区域，这些识别区域将靶向特征性细胞表面标记物;（2）用于在识别区域之间建立通信的寡核苷酸;以及（3）在抗体交联时触发药物释放的药物递送组件。作为我们的三个目标，我们将在未来三年内测试在细胞上实现三种不同程序（或布尔逻辑运算）的混合物：1。{M1ANDM 2凋亡};一种三组分自动机，其作用将导致抗体的交联和仅在展示用于靶向B细胞淋巴瘤系的两种细胞表面标记的细胞中的凋亡的显著增加。{M1ANDM 2药物释放};另一个三组件自动机，类似于上面的一个，除了它也会触发一个小的细胞毒性分子的释放。{M3 ANDNOTM 1 Drug Release}，一个三分量自动机，它将考虑一个保护因子。智能分子自动机是分子网络，根据它们识别的输入和它们实现的算法的存在或不存在来改变状态。在这个建议中，我们研究了下一代的分子自动机能够分析多种细胞表面或疾病标志物。据我们所知，用抗体与寡核苷酸结合来构建布尔自动机的提议是一个完全新颖的想法。更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。所有基于靶向消除细胞亚群（例如癌症、自身免疫性疾病）的治疗都将受益于考虑一种以上细胞表面标记物的能力，因为很少有细胞表面标记物仅限于一种细胞类型。教育意义在教育方面，我们有一个非常富有成效的计划，将当地高中学生融入我们的团队。一个学生从这个程序已经最近的第一作者出版物描述分子全加器，和几个学生将共同作者的手稿，我们正在准备复杂的自动机。在这个项目中，我们计划在第一年让一名暑期学生参与针对一种细胞表面标记的适体的分离。第二个学生将在体外优化的非门操作所需的结构切换过程中，在这个程序的第二年。他们将由一名学术型博士后进行培训。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.