Milo Lin, a physicist and Assistant Professor in the Department of Bioinformatics at the University of Texas Southwestern Medical Center, has developed a "mapping” that, when combined with thermodynamic circuit theory, allows one to transform a biochemical network into an equivalent electrical circuit that obeys Ohm’s law. This allows the theorems and powerful quantitative methods of electrical-network-analysis developed over the past century to be applied to biochemical networks. These electrical engineering tools have -for the electronics industry- enabled large-scale system prediction and design through abstraction and modularity as opposed to simulation of all the components and interactions in a given system. Funds from this grant support efforts by Professor Lin to deploy this framework in the analysis of biochemical systems. Lin will begin by systematically mapping a wide variety of biochemical networks found in organisms (regulatory networks, metabolic pathways, molecular motors, etc.) to equivalent electric circuits and then using electrical engineering tools to obtain the simplest circuit of that type. He will then use computer simulations to “evolve” this simple circuit to meet various targets, including determining circuits necessary and sufficient to execute arbitrary computations, circuits that exhibit robustness to input noise, and circuits that exhibit robustness to changes in the fitness landscape.  Lin will then explore the possible existence of a threshold thermodynamic force above which biomolecular computational capacity is dramatically increased, which, if there is a such a threshold, may shed some light on the puzzling observation that living systems overwhelmingly choose nonequilibrium over equilibrium chemistry for computation. If successful, Lin’s project will facilitate our understanding of complex biochemical networks and therefore of how organisms use chemistry to achieve life-sustaining functions. More speculatively, characterizing the computational capacity of a wide range of biochemical networks may provide insights that allow one to delineate living from nonliving matter.

This grant supports research by Chunte (Sam) Peng, an Assistant Professor of Chemistry at Massachusetts Institute of Technology (MIT) and a member of the MIT-Harvard Broad Institute, which will focus on creating novel probes for single particle tracking to quantitatively study the nonequilibrium dynamics of molecular motors in vivo, using these dynamics as a window into the functioning of living systems and far-from-equilibrium physics. Professor Peng proposes to learn about intracellular dynamics by making movies of molecular motors ‘tagged’ with fluorescent probes. The probes—called Up Conversion NanoParticles (UCNPs)—have three features that make them superior to existing fluorescent probes and which suit them for intracellular single particle tracking. First, UCNPs fluoresce at different colors than the light emitted by other cellular components, thus offering superior contrast relative to nearby objects. Second, UCNP fluorescence is spectrally much narrower than that of commonly used fluorescent probes, allowing a larger number of distinct cellular components—each tagged with a different color—to be simultaneously tracked. Third, UCNPs can be used to track a cellular object for much longer (hours or days) than is typical using existing fluorescent probes (a few minutes). Peng will use UCNPs to study the nonequilibrium dynamics of two molecular motors, dynein and kinesin, as they transport ‘cargo’ from place to place within a cell along intracellular protein polymers. The research team will attach probes to cargo and/or motors and then record movies that track probe position as revealed by the UCNP fluorescence. The plan is to begin by quantifying motor dynamics at various points in a motor’s traversal of a cell and then explore why motor behavior varies by gaining an ever-increasing level of detail about the local cellular environment. Particular experimental attention will be paid to how motor efficiency varies in relation to varying cellular conditions, efficiency-affecting interactions between motors and between motors and cargo, and the relation of observed motor efficiency to efficiency constraints predicted by thermodynamic theory. 

Multicellular organisms operate through cell differentiation.  What begins as an initial pool of uniform, isogenic cells eventually develops into a suite of distinct cell types, each performing specialized functions in the organism as a whole. This specialization process can be thought of as a form of information processing . A simplified overall picture of this information processing is that a receptor -for instance at the cell membrane- detects a signal molecule and a signaling network then acts as 'wiring' to relay information to downstream components, which then produce an appropriate cellular response. The signaling network is a chain of biochemical events which link an upstream molecular signal to a downstream 'cis-regulation' system in order to achieve a particular gene-expression pattern that--in this case--specifies cell type.  This grant supports work by Michael Elowitz, Professor of Biology and of Bioengineering at Caltech, to create a synthetic cell fate control system. The system, if successful, will allow one to begin with a pool of genetically identical cells and then -using a small number of 'input' signals- direct various subsets of the pool to differentiate and develop into distinct, predetermined cell types. Elowitz proposes to achieve this goal by using the tools of synthetic biology to develop the two core subsystems briefly sketched above: a 'signaling network'; a complex network of proteins to process chemical 'signals' and relay them downstream and a downstream 'cis-regulation' system that drives expression of a specific gene or set of genes. The signaling network and gene regulation system will be integrated into natural cells to demonstrate cell fate control and differentiation into targeted cell types. If successful, this project will advance our understanding of and control over cellular information processing and provide a foundation for extending synthetic biology into multicellularity.

Understanding how matter complexifies, ultimately towards life, is a longstanding challenge embraced by the Matter-to-Life program. Since life as we know it is primarily chemistry, the challenge amounts to understanding how complexity and function can emerge and grow in a complicated chemical network. This grant supports Leroy Cronin, Professor of Chemistry at the University of Glasgow, to deploy a systems chemistry approach to addressing this question. Cronin will leverage state-of-the-art robotics to enable long-term chemical evolution experiments that exploit a new parameter that quantifies molecular complexity that is both experimentally accessible and embedded within a larger theoretical framework. Cronin plans to use that framework to nudge a chemical system towards ever-increasing complexity. Selection as a concept is most commonly deployed within Darwinian evolution, where natural selection refers to the preferential survival of individuals with certain genetic traits by means of natural controlling factors. Here Professor Cronin proposes to observe selection within a chemical system, and in this context, selection refers to the preferential survival of molecules with certain traits by means of natural controlling factors (the local environment). Cronin is primarily interested in one trait: complexity. Professor Cronin will use a measure of molecular complexity called the “assembly index.” The assembly index of a molecule is, in essence, equal to the number of ‘steps’ (chemical bonds) needed to construct the molecule using system-dependent basic building blocks (atoms or molecules).  Cronin has demonstrated that assembly index is well-correlated to three relatively-easy-to-perform types of measurements: mass spectrometry, IR spectroscopy, and NMR spectroscopy. The research team will run recursive chemistry experiments that rely on automated measurements to determine which molecules are present and adjust conditions to nudge a system towards a ‘selection regime’ where new forms of complexity are generated. The adjustable conditions include things like temperature (heating & cooling), evaporation and rehydration, how long a mixture is stirred, the duration of an experimental cycle, solution pH, whether various minerals are added, and whether or not an electrical discharge is applied. Cronin expects that a single experiment (a series of cycles) will run continuously for several hundred cycles, corresponding to several weeks or months. Over that time, there are four types of complex molecules / structures that the researchers will seek to detect: self-reproducing molecules and autocatalytic sets; production of high assembly index molecules; formation of primitive sequence polymers; and emergence of microscopic containers.

Training the next generation of researchers is an essential component of any healthy academic field. Here William Bialek and Joshua Shaevitz, Professors of Physics at Princeton University and Co-Directors of the Center for the Physics of Biological Function, request three years of support for a Center Fellow pursuing physics-of-life research. This prestigious postdoctoral fellowship will offer a young researcher both intellectual freedom and a support structure, and grant funds would support either a theorist or an experimentalist. A fellowship offering intellectual freedom to an early-career scholar is typically challenging to fund through federal agencies focused on supporting specific projects, despite the fact that this freedom can play an important role in establishing a young scientist as an independent researcher. The Center for the Physics of Biological Function is a partnership between Princeton and the Graduate Center of the City University of New York; a partnership anchored by a core community of sixteen CUNY/Princeton faculty. The Center focuses on science at the interface of physics and biology with the goal of creating ‘a physicist’s understanding of living systems: a physics of biological function that connects the myriad details of life, across all scales, to fundamental and universal physical principles.’ Center Fellows will be offered a competitive salary, travel funds, and independence to select a compelling line of research. The Center Fellow is not obligated to any particular faculty member, instead the Center exposes young physicists to problems posed by a wide range of living systems and gives them ‘considerable freedom to explore these problems, crossing boundaries among topics that would be in separate groups or departments at most institutions.’ This freedom is balanced by a support system as the Fellow is held accountable to formulating a feasible plan by interacting with senior Center faculty, and there’s a community of Fellows that provide peer advice and guidance.

Systems chemistry is a branch of chemical research that examines large, complicated chemical networks and focuses on understanding how complexity and function can emerge from the many diverse chemical interactions within the network. This grant provides support to a team led by Rein Ulijn, Professor of Physics and Director of the Nanoscience Initiative at the City University of New York Graduate Center, to induce a chemical system to demonstrate a life-like behavior; specifically, a primitive form of learning and memory. Dr. Ulijn’s basic chemical system will be composed of peptides (short proteins; a sequence of amino acids) that -with the help of an enzyme- can be reversibly combined into more complex peptides (oligopeptides). The team plans to expose a chemical network to a molecule that acts as an environmental stimulus that will cause a reaction, the formation of new peptide molecules and various phase-separated peptide ‘structures.’ After removing the stimulus molecules from the network, they will be re-introducing at some later time.  If the network responds more rapidly to the stimulus than when the molecules were first introduced, it has, in a basic sense, “remembered” the initial stimulus and ‘learned’ to respond faster.  The project begins by choosing an initial set of (2-6) interacting dipeptides. Molecular dynamics simulations will inform selection of the initial dipeptide system to ensure that the dipeptides have a propensity for self-assembly. This makes it likely that more complex peptides and (peptide) structures will form. Once an initial system has been selected, the researchers will synthesize the system in their lab, allow the peptide chemistry to run to a steady state, and then characterize the steady state distribution of peptides and phase-separated structures in the unperturbed system (i.e. before a stimulus molecule is introduced). The formation of oligopeptides and phase-separated structures will be monitored using a combination of microscopy, optical spectroscopy, liquid chromatography, mass spectrometry, and dynamic light scattering. Once the steady state properties of the unperturbed peptide system have been characterized, a stimulus molecule will be introduced and the researchers will characterize how the distribution of peptides and the formation of structures is modified. The characterization will be done for each of several stimulus molecules (flavor molecules grape, raspberry, banana and apple) selected based on their simplicity and because they offer a systematic variation in chemical-interaction potential.  Finally, the researchers will determine whether repeated exposure to a given stimulus molecule can condition the system to respond more rapidly; the stimulus molecules will be removed between exposures. The researchers will study how learning and memory are influenced by variation of experimental parameters such as pH, temperature, and molecular target concentration. They’ll also test the hypothesis that the physical basis of memory lies in remnant structures retained by the solution. This idea will be tested by using heat to melt any remnant structural nuclei; something that should eliminate any observed memory effects. Beyond demonstration of learning and memory induced by a single type of stimulus molecule, the researchers will also explore exposure to competing stimuli, by examining whether mixing of separately conditioned solutions yields a different response when compared to that obtained from a solution conditioned by simultaneous exposure to several types of stimulus molecules.

Earth biochemistry relies on DNA as the information-carrying polymer and water as the chemistry-facilitating solvent. Life on other planets, however, could leverage very different chemistry. This grant supports work by Sara Seager, Professor of Planetary Science, Physics, and Aeronautical and Astronautical Engineering at the Massachusetts Institute of Technology, that will explore an alternative to Earth biochemistry. The proposed research focuses on identifying a DNA-like molecule that is functional in concentrated sulfuric acid (CSA), the primary component of Venus’ atmosphere and a water-alternative solvent found on many planets in our galaxy.   There are several steps to establishing that a DNA-like molecule can function in CSA, and Professor Seager is tackling what is perhaps the core challenge: identifying components of a DNA-analog molecule that are structurally stable and appropriately reactive in CSA, focusing on the three primary molecular components of DNA: nucleic acid bases, so-called ‘linker’ molecules, and a ‘molecular backbone’ structure. Her project is divided into four tasks. In Task 1 Seager and her researcher team will determine the CSA reactivity of the nucleic acid bases found in DNA/RNA. While Seager has demonstrated that the core structures of these canonical bases are CSA-stable, it’s not yet known whether the bases can bond with one another in CSA; something required to form a DNA-like molecule.   Excess protons found in CSA (or in any acid) may interfere with the hydrogen bonding that holds two bases together in a DNA molecule, making base-pairing with these canonical bases impossible in CSA. Accordingly, in Task 2 the researcher team will test the CSA stability and reactivity of ‘alternative’ nucleic acid bases that do not rely on hydrogen bonding for base-pairing. In Task 3, the researchers will develop a list of linker and backbone molecule candidates that promise to be stable in CSA and in Task 4 these candidates will be subjected to CSA stability/reactivity testing.   Establishing that a replicating, information-bearing molecule can exist in CSA goes a long way to establishing CSA as a solvent that can host life. Such a finding would significantly impact exoplanet research, expand the number of planets regarded as habitable, and inform planned and proposed missions to Venus aimed at searching for signs of extraterrestrial life.

Life as we know it is primarily chemistry: all living organisms are composed of carbon-based molecules such as proteins, carbohydrates, and nucleic acids that are the result of chemical reactions between different elements like carbon, hydrogen, oxygen, nitrogen, and sulfur. Understanding the basic principles underlying chemical reactions is important to understanding life. When thinking about reactions in a chemical context, scientists typically think about reactants interacting in a homogeneous solution that’s essentially the same in any given place. But the cytoplasm, the liquid in cells where biochemical reactions take place, is a biological context that’s quite different from the simplified models of chemistry textbooks. Cytoplasm is a heterogeneous fluid that looks and functions differently across the cell and so it’s not accurately represented by simplifying assumptions of homogeneity. Precisely how these assumptions are distorting our picture of cellular chemistry, and therefore our understanding of fundamental biochemistry, is not well understood. This grant supports Lauren Zarzar and Ayusman Sen at the Pennsylvania State University who will study how solution heterogeneity influences reactivity for three important classes of biochemical reactions. First, they will study autocatalytic reactions, in which one of the reaction products facilitates (i.e. is a catalyst for) the same, or a coupled, reaction. Autocatalysis is a mechanism for chemical self-replication and is considered a key aspect of the prebiotic chemistry that gave rise to life. Next, the team will study enzyme reaction cascades, a sequence of enzyme-catalyzed reactions whereby the product of one reaction is the reactant for the next reaction. They’ll focus on chemotaxis (chemical activity that leads to motion towards or away from a higher concentration of some substance) to study how solution structure affects enzyme cascades. Finally, they will study polymerization reactions and, in doing so, address an important question in prebiotic chemistry: how polymers with specific monomer sequences arise without a specific sequence-directing mechanism. Ultimately, this project will deepen our understanding of specific reactions central to cellular chemistry and shed light on the role solution heterogeneity plays in driving the chemistry of life.