Synthetic molecular machines for active self-assembly : prototype algorithms, designs, and experimental study

Computer science and electrical engineering have been the great success story of the twentieth century. The neat modularity and mapping of a language onto circuits has led to robots on Mars, desktop computers and smartphones. But these devices are not yet able to do some of the things that life takes for granted: repair a scratch, reproduce, regenerate, or grow exponentially fast–all while remaining functional.

This thesis explores and develops algorithms, molecular implementations, and theoretical proofs in the context of “active self-assembly” of molecular systems. The long-term vision of active self-assembly is the theoretical and physical implementation of materials that are composed of reconfigurable units with the programmability and adaptability of biology’s numerous molecular machines. En route to this goal, we must first find a way to overcome the memory limitations of molecular systems, and to discover the limits of complexity that can be achieved with individual molecules.

One of the main thrusts in molecular programming is to use computer science as a tool for figuring out what can be achieved. While molecular systems that are Turing-complete have been demonstrated [Winfree, 1996], these systems still cannot achieve some of the feats biology has achieved.

One might think that because a system is Turing-complete, capable of computing “anything,” that it can do any arbitrary task. But while it can simulate any digital computational problem, there are many behaviors that are not “computations” in a classical sense, and cannot be directly implemented. Examples include exponential growth and molecular motion relative to a surface.

Passive self-assembly systems cannot implement these behaviors because (a) molecular motion relative to a surface requires a source of fuel that is external to the system, and (b) passive systems are too slow to assemble exponentially-fast-growing structures. We call these behaviors “energetically incomplete” programmable behaviors. This class of behaviors includes any behavior where a passive physical system simply does not have enough physical energy to perform the specified tasks in the requisite amount of time.

As we will demonstrate and prove, a sufficiently expressive implementation of an “active” molecular self-assembly approach can achieve these behaviors. Using an external source of fuel solves part of the the problem, so the system is not “energetically incomplete.” But the programmable system also needs to have sufficient expressive power to achieve the specified behaviors. Perhaps surprisingly, some of these systems do not even require Turing completeness to be sufficiently expressive.

Building on a large variety of work by other scientists in the fields of DNA nanotechnology, chemistry and reconfigurable robotics, this thesis introduces several research contributions in the context of active self-assembly.

We show that simple primitives such as insertion and deletion are able to generate complex and interesting results such as the growth of a linear polymer in logarithmic time and the ability of a linear polymer to treadmill. To this end we developed a formal model for active-self assembly that is directly implementable with DNA molecules. We show that this model is computationally equivalent to a machine capable of producing strings that are stronger than regular languages and, at most, as strong as context-free grammars. This is a great advance in the theory of active self- assembly as prior models were either entirely theoretical or only implementable in the context of macro-scale robotics.

We developed a chain reaction method for the autonomous exponential growth of a linear DNA polymer. Our method is based on the insertion of molecules into the assembly, which generates two new insertion sites for every initial one employed. The building of a line in logarithmic time is a first step toward building a shape in logarithmic time. We demonstrate the first construction of a synthetic linear polymer that grows exponentially fast via insertion. We show that monomer molecules are converted into the polymer in logarithmic time via spectrofluorimetry and gel electrophoresis experiments. We also demonstrate the division of these polymers via the addition of a single DNA complex that competes with the insertion mechanism. This shows the growth of a population of polymers in logarithmic time. We characterize the DNA insertion mechanism that we utilize in Chapter 4. We experimentally demonstrate that we can control the kinetics of this re- action over at least seven orders of magnitude, by programming the sequences of DNA that initiate the reaction.

In addition, we review co-authored work on programming molecular robots using prescriptive landscapes of DNA origami; this was the first microscopic demonstration of programming a molec- ular robot to walk on a 2-dimensional surface. We developed a snapshot method for imaging these random walking molecular robots and a CAPTCHA-like analysis method for difficult-to-interpret imaging data.

Equilibrium in dynamic economic models

The main theme running through these three chapters is that economic agents are often forced to respond to events that are not a direct result of their actions or other agents actions. The optimal response to these shocks will necessarily depend on agents' understanding of how these shocks arise. The economic environment in the first two chapters is analogous to the classic chain store game. In this setting, the addition of unintended trembles by the agents creates an environment better suited to reputation building. The third chapter considers the competitive equilibrium price dynamics in an overlapping generations environment when there are supply and demand shocks.

The first chapter is a game theoretic investigation of a reputation building game. A sequential equilibrium model, called the "error prone agents" model, is developed. In this model, agents believe that all actions are potentially subjected to an error process. Inclusion of this belief into the equilibrium calculation provides for a richer class of reputation building possibilities than when perfect implementation is assumed.

In the second chapter, maximum likelihood estimation is employed to test the consistency of this new model and other models with data from experiments run by other researchers that served as the basis for prominent papers in this field. The alternate models considered are essentially modifications to the standard sequential equilibrium. While some models perform quite well in that the nature of the modification seems to explain deviations from the sequential equilibrium quite well, the degree to which these modifications must be applied shows no consistency across different experimental designs.

The third chapter is a study of price dynamics in an overlapping generations model. It establishes the existence of a unique perfect-foresight competitive equilibrium price path in a pure exchange economy with a finite time horizon when there are arbitrarily many shocks to supply or demand. One main reason for the interest in this equilibrium is that overlapping generations environments are very fruitful for the study of price dynamics, especially in experimental settings. The perfect foresight assumption is an important place to start when examining these environments because it will produce the ex post socially efficient allocation of goods. This characteristic makes this a natural baseline to which other models of price dynamics could be compared.

Signal transduction with hybridization chain reactions

Some of the most exciting developments in the field of nucleic acid engineering include the utilization of synthetic nucleic acid molecular devices as gene regulators, as disease marker detectors, and most recently, as therapeutic agents. The common thread between these technologies is their reliance on the detection of specific nucleic acid input markers to generate some desirable output, such as a change in the copy number of an mRNA (for gene regulation), a change in the emitted light intensity (for some diagnostics), and a change in cell state within an organism (for therapeutics). The research presented in this thesis likewise focuses on engineering molecular tools that detect specific nucleic acid inputs, and respond with useful outputs.

Four contributions to the field of nucleic acid engineering are presented: (1) the construction of a single nucleotide polymorphism (SNP) detector based on the mechanism of hybridization chain reaction (HCR); (2) the utilization of a single-stranded oligonucleotide molecular Scavenger as a means of enhancing HCR selectivity; (3) the implementation of Quenched HCR, a technique that facilitates transduction of a nucleic acid chemical input into an optical (light) output, and (4) the engineering of conditional probes that function as sequence transducers, receiving target signal as input and providing a sequence of choice as output. These programmable molecular systems are conceptually well-suited for performing wash-free, highly selective rapid genotyping and expression profiling in vitro, in situ, and potentially in living cells.

An arithmetical theorem for partially ordered sets

The simplest multiplicative systems in which arithmetical ideas can be defined are semigroups. For such systems irreducible (prime) elements can be introduced and conditions under which the fundamental theorem of arithmetic holds have been investigated (Clifford (3)). After identifying associates, the elements of the semigroup form a partially ordered set with respect to the ordinary division relation. This suggests the possibility of an analogous arithmetical result for abstract partially ordered sets. Although nothing corresponding to product exists in a partially ordered set, there is a notion similar to g.c.d. This is the meet operation, defined as greatest lower bound. Thus irreducible elements, namely those elements not expressible as meets of proper divisors can be introduced. The assumption of the ascending chain condition then implies that each element is representable as a reduced meet of irreducibles. The central problem of this thesis is to determine conditions on the structure of the partially ordered set in order that each element have a unique such representation.

Part I contains preliminary results and introduces the principal tools of the investigation. In the second part, basic properties of the lattice of ideals and the connection between its structure and the irreducible decompositions of elements are developed. The proofs of these results are identical with the corresponding ones for the lattice case (Dilworth (2)). The last part contains those results whose proofs are peculiar to partially ordered sets and also contains the proof of the main theorem.

Chain-forming zintl antimonides as novel thermoelectric materials

Zintl phases, a subset of intermetallic compounds characterized by covalently-bonded "sub-structures," surrounded by highly electropositive cations, exhibit precisely the characteristics desired for thermoelectric applications. The requirement that Zintl compounds satisfy the valence of anions through the formation of covalent substructures leads to many unique, complex crystal structures. Such complexity often leads to exceptionally low lattice thermal conductivity due to the containment of heat in low velocity optical modes in the phonon dispersion. To date, excellent thermoelectric properties have been demonstrated in several Zintl compounds. However, compared with the large number of known Zintl phases, very few have been investigated as thermoelectric materials.

From this pool of uninvestigated compounds, we selected a class of Zintl antimonides that share a common structural motif: anionic moieties resembling infinite chains of linked MSb₄ tetrahedra, where $M$ is a triel element. The compounds discussed in this thesis (A₅M₂Sb₆ and A₃MSb₃, where A = Ca or Sr and M = Al, Ga and In) crystallize as four distinct, but closely related "chain-forming" structure types. This thesis describes the thermoelectric characterization and optimization of these phases, and explores the influence of their chemistry and structure on the thermal and electronic transport properties. Due to their large unit cells, each compound exhibits exceptionally low lattice thermal conductivity (0.4 - 0.6 W/mK at 1000 K), approaching the predicted glassy minimum at high temperatures. A combination of Density Functional calculations and classical transport models were used to explain the experimentally observed electronic transport properties of each compound. Consistent with the Zintl electron counting formalism, A₅M₂Sb₆ and A₃MSb₃ phases were found to have filled valence bands and exhibit intrinsic electronic properties. Doping with divalent transition metals (Zn²⁺ and Mn²⁺) on the M³⁺ site, or Na¹⁺ on the A³⁺ site allowed for rational control of the carrier concentration and a transition towards degenerate semiconducting behavior. In optimally-doped samples, promising peak zT values between 0.4 and 0.9 were obtained, highlighting the value of continued investigations of complex Zintl phases.

Investigation of Fundamental Processes Governing Secondary Organic Aerosol Formation in Laboratory Chambers

Our understanding of the processes and mechanisms by which secondary organic aerosol (SOA) is formed is derived from laboratory chamber studies. In the atmosphere, SOA formation is primarily driven by progressive photooxidation of SOA precursors, coupled with their gas-particle partitioning. In the chamber environment, SOA-forming vapors undergo multiple chemical and physical processes that involve production and removal via gas-phase reactions; partitioning onto suspended particles vs. particles deposited on the chamber wall; and direct deposition on the chamber wall. The main focus of this dissertation is to characterize the interactions of organic vapors with suspended particles and the chamber wall and explore how these intertwined processes in laboratory chambers govern SOA formation and evolution.

A Functional Group Oxidation Model (FGOM) that represents SOA formation and evolution in terms of the competition between functionalization and fragmentation, the extent of oxygen atom addition, and the change of volatility, is developed. The FGOM contains a set of parameters that are to be determined by fitting of the model to laboratory chamber data. The sensitivity of the model prediction to variation of the adjustable parameters allows one to assess the relative importance of various pathways involved in SOA formation.

A critical aspect of the environmental chamber is the presence of the wall, which can induce deposition of SOA-forming vapors and promote heterogeneous reactions. An experimental protocol and model framework are first developed to constrain the vapor-wall interactions. By optimal fitting the model predictions to the observed wall-induced decay profiles of 25 oxidized organic compounds, the dominant parameter governing the extent of wall deposition of a compound is identified, i.e., wall accommodation coefficient. By correlating this parameter with the molecular properties of a compound via its volatility, the wall-induced deposition rate of an organic compound can be predicted based on its carbon and oxygen numbers in the molecule.

Heterogeneous transformation of δ-hydroxycarbonyl, a major first-generation product from long-chain alkane photochemistry, is observed on the surface of particles and walls. The uniqueness of this reaction scheme is the production of substituted dihydrofuran, which is highly reactive towards ozone, OH, and NO3, thereby opening a reaction pathway that is not usually accessible to alkanes. A spectrum of highly-oxygenated products with carboxylic acid, ester, and ether functional groups is produced from the substituted dihydrofuran chemistry, thereby affecting the average oxidation state of the alkane-derived SOA.

The vapor wall loss correction is applied to several chamber-derived SOA systems generated from both anthropogenic and biogenic sources. Experimental and modeling approaches are employed to constrain the partitioning behavior of SOA-forming vapors onto suspended particles vs. chamber walls. It is demonstrated that deposition of SOA-forming vapors to the chamber wall during photooxidation experiments can lead to substantial and systematic underestimation of SOA. Therefore, it is likely that a lack of proper accounting for vapor wall losses that suppress chamber-derived SOA yields contribute substantially to the underprediction of ambient SOA concentrations in atmospheric models.

Stationary absolute distributions for chains of infinite order

Let {Ƶ_n}^∞_{n = -∞} be a stochastic process with state space S₁ = {0, 1, …, D – 1}. Such a process is called a chain of infinite order. The transitions of the chain are described by the functions

Q_i(i⁽⁰⁾) = Ƥ(Ƶ_n = i | Ƶ_{n - 1} = i ⁽⁰⁾₁, Ƶ_{n - 2} = i ⁽⁰⁾₂, …) (i ɛ S₁), where i⁽⁰⁾ = (i⁽⁰⁾₁, i⁽⁰⁾₂, …) ranges over infinite sequences from S₁. If i⁽ⁿ⁾ = (i⁽ⁿ⁾₁, i⁽ⁿ⁾₂, …) for n = 1, 2,…, then i⁽ⁿ⁾ → i⁽⁰⁾ means that for each k, i⁽ⁿ⁾_k = i⁽⁰⁾_k for all n sufficiently large.

Given functions Q_i(i⁽⁰⁾) such that

(i) 0 ≤ Q_i(i⁽⁰) ≤ ξ ˂ 1

(ii)D – 1/Ʃ/i = 0 Q_i(i⁽⁰⁾) Ξ 1

(iii) Q_i(i⁽ⁿ⁾) → Q_i(i⁽⁰⁾) whenever i⁽ⁿ⁾ → i⁽⁰⁾,

we prove the existence of a stationary chain of infinite order {Ƶ_n} whose transitions are given by

Ƥ (Ƶ_n = i | Ƶ_{n - 1}, Ƶ_{n - 2}, …) = Q_i(Ƶ_{n - 1}, Ƶ_{n - 2}, …)

With probability 1. The method also yields stationary chains {Ƶ_n} for which (iii) does not hold but whose transition probabilities are, in a sense, “locally Markovian.” These and similar results extend a paper by T.E. Harris [Pac. J. Math., 5 (1955), 707-724].

Included is a new proof of the existence and uniqueness of a stationary absolute distribution for an Nth order Markov chain in which all transitions are possible. This proof allows us to achieve our main results without the use of limit theorem techniques.