EMT: Cell Death by Boolean Calculations with Antibodies

未来三年，我们的目标是证明人工合成分子的混合物，我们称之为治疗性分子自动机，可以在细胞表面进行布尔计算，并产生治疗性结果（即细胞死亡）。具体来说，我们将探索分子自动机的一种特定亚型，由基于抗体的组件的混合物组成，这些组件将组装在细胞表面上，根据细胞表面制造商（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，将针对特征细胞表面制造商；(2)用于在识别区域之间建立通信的寡核苷酸；(3)在抗体交联后触发药物释放的药物传递组件。作为我们的三个目标，我们将在未来三年内测试在单元上实现三种不同程序（或布尔逻辑操作）的混合物：1。{M1ANDM2凋亡};一种三组分自动机，其作用将导致抗体交联和凋亡的显著增加，仅在具有用于靶向b细胞淋巴瘤系的两种细胞表面标记的细胞中。{M1ANDM2药物释放}；另一个三组分自动机，类似于上面的一个，除了它也会触发一个小的细胞毒性分子的释放。{M3ANDNOTM1药物释放}，一个三组分自动机，它将考虑一个保护因素。分子自动机是由分子组成的网络，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。在本提案中，我们研究能够分析多种细胞表面或疾病标记的下一代分子自动机。据我们所知，用偶联抗体构建布尔自动机是一个完全新颖的想法。更广泛的意义生物医学意义：我们的自动机有潜力在广泛的疾病过程中发挥作用。所有基于靶向消除细胞亚群（例如癌症、自身免疫性疾病）的疗法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。教育的意义。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小组。这个项目的一名学生最近在一篇描述分子全加法器的出版物中成为第一作者，还有几名学生将成为我们正在准备的关于复杂自动机的手稿的共同作者。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物的适体分离。第二名学生将在体外优化非栅极操作所需的结构切换过程。他们将由一个以学术为导向的博士后进行培训。跨学科的方法。我们注意到这个项目的跨学科性质，它结合了纳米技术、电子工程和计算机科学、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.