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 merit Molecular 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 Significance Biomedical 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.
