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Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function via Dynamic RNA Nanotechnology
Get PDFA guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. There are two possible logical directions for single-input cgRNAs: constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON→OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF→ON logic). Using an in vitro assay for cgRNA activity with synthetic trigger, in vitro transcribed cgRNA, and recombinant dCas9, we observe a conditional (ON→OFF logic) response for a set of four allosteric constitutively active cgRNAs with a median ≈6% crosstalk between noncognate cgRNA/trigger pairs. Motivated by the observed lack of conditional response of this mechanism when ported to E. coli, we describe a systematic study of unstructured sequence inserts into the standard gRNA structure and report the conditional response of a set of 34 candidate cgRNAs in living cells. Molecular mechanisms for both ON→OFF and OFF→ON cgRNAs are demonstrated in E. coli. For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to produce an orthogonal library of cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈15-fold for a library of three orthogonal ON→OFF "splinted switch" cgRNA/trigger pairs, and ≈3-fold for a library of three orthogonal OFF→ON "toehold switch" cgRNA/trigger pairs; the median crosstalk within each library is <2% and ≈20% for the two mechanisms, respectively. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for conditional gene regulation and synthetic biology.</p
Investigation of Ethylene Tetramerization Catalysis from Structurally-Defined Organochromium Compounds
Get PDFChapter 1 is a general introduction to the topic of ethylene tetramerization catalysis.
Chapter 2 presents the synthesis and catalytic utility of chromium multi-aryl complexes that were the first examples of ethylene tetramerization catalysts that could be produced without excess alkyl aluminum reagents.
Chapter 3 describes the mechanistic analysis of the ethylene tetramerization reaction using isotopically labelled ethylene. Co-production of 1-hexene along with 1-octene was determined to be intrinsic to the reaction mechanism. This is due to the intermediacy of a chromacyclic species that can either eliminate 1-hexene or insert a fourth ethylene.
Chapter 4 presents the synthesis of additional Cr tris(aryl) complexes, which are coordinatively saturated, and were used to generate a crystallographically-characterized Cr(III) cationic species. This was the first reported single-component precatalyst for ethylene tetramerization.
Chapter 5 describes the isotopic labelling of a well-defined ethylene tetramerization precatalyst with a deuteriomethyl group. This label was tracked following protonation of the neutral Cr complex via pulse EPR. Successful detection of deuterium on Cr-alkyl ligands led to in situ analysis of the catalytic mixture. A low-spin species derived from deuterated ethylene was observed.
Appendix 1 describes the synthesis of various Cr aryl amine complexes. Appendix 2 provides the results of additional catalytic experiments for ethylene tetramerization, including those with a more soluble precatalyst, and those at higher ethylene pressure. Appendix 3 details the synthesis of a molecular Re catalyst for CO2 electroreduction which was used to modify electrodes. Appendix 4 lists various X-ray crystal structures that were obtained, but not related elsewhere in the thesis.</p
Resistance is Futile: Physical Science, Systems Biology and Single-Cell Analysis to Understanding the Plastic and Heterogeneous Nature of Melanoma and Their Role in Non-Genetic Drug Resistance
Get PDFMelanoma is the most deadly form of skin cancer due to its great metastatic potential. Targeted therapy that inhibits the BRAF-V600E driver mutation has shown impressive initial responses in melanoma patients. However, drug resistance, as the universal phenomenon for any cancer therapy, always limits treatment efficacy and compromises outcomes. As the early-step of resistance development, non-genetic mechanisms enable cancer cells to transition into a drug-resistant state in as early as a few days after drug treatment without alteration of the genome. This early mechanism is, to a large extent, due to the heterogeneous and highly plastic nature of tumor cells. Therefore, it imperative to understand the plastic and heterogeneous nature of the melanoma cells in order to identify combination therapies that can overcome resistance.
In this thesis, we investigate these two fundamental natures of non-genetic drug resistance using BRAF inhibition of BRAF-mutant melanomas as the model system. These melanoma cells undergo multi-step, reversible drug-induced cell-state transitions from the original sensitive phenotype to a drug-resistant one.
We first conducted bulk analysis to characterize the detailed kinetics of the entire transition from drug-sensitive state towards drug-resistant state, revealing expression changes of thousands of genes and extensive chromatin remodeling. A 3-step computational biology approach greatly simplified the complexity and revealed that the whole cell-state transition was controlled by a gene module activated within just the first three days of drug treatment, with the RelA transcription factor driving chromatin remodeling to establish an epigenetic program encoding long-term phenotype changes towards resistance. From there, a detailed mechanism connecting tumor epigenetic plasticity with non-genetic drug resistance was resolved through in-depth molecular biology experiments. The mechanism was validated in clinical patient samples.
We further investigated heterogeneity by moving from bulk cellular studies to single-cell analysis. The single-cell view further revealed that two driving forces from both cell-state interconversions and phenotype-specific drug selection control the cell-state transition dynamics. The single-cell studies also pinpointed the signaling network hub, RelA, as the driver molecule of the initiation of the adaptive transition. These two competing driving forces were further quantitatively modeled via a thermodynamic-inspired surprisal analysis and a modified Fokker-Planck-type kinetic model.
Finally, using integrated single-cell proteomic and metabolic technology I developed to characterize the early-stage signaling and metabolic changes upon initial drug responses, we further identified two distinct paths connecting drug-sensitive and drug-tolerant states. Melanoma cells exclusively traverse one of the two paths depending on the level of MITF in the drug-naïve cells. The two trajectories are associated with distinct signaling and metabolic susceptibilities and are independently druggable.
In total, this thesis combines and synergizes various physical science and systems biology approaches together with several unique single-cell technologies and analysis to obtain a deep and comprehensive understanding of non-genetic drug resistance in cancer. The findings from this thesis provide several novel insights into the rational design of effective combination therapy for overcoming the development of resistance in response to cancer treatments.</p
Probing the Nature of Black Holes with Gravitational Waves
Get PDFIn this thesis, I present a number of studies intended to improve our understanding of black holes using gravitational waves. Although black holes are relatively well understood from a theory perspective, many questions remain about the nature of the black holes in our Universe. According to general relativity, astrophysical black holes are fully described by just their mass and spin. Yet, relying on electromagnetic-based observatories alone, we still know very little about the distribution of black hole masses or spins. Moreover, as merging black holes are invisible to these electromagnetic observatories, we cannot rely on them to provide us with information about the binary black hole merger rate or binary black hole formation channels. However, by observing gravitational wave signals from these inherently dark binaries, we will soon have some answers to these questions. Indeed, the Laser Interferometer Gravitational-Wave Observatory (LIGO) has already revealed a great deal of new information about binary black holes; giving us an early glimpse into their mass and spin distributions and placing the first constraints on the binary black hole merger rate. This thesis contributes to the goal of probing the nature of black holes with gravitational waves.
Binary black holes can form as an isolated binary in the galactic field or through dynamical encounters in high-density environments. Dynamical formation can significantly alter the binary parameters, which then become imprinted on the gravitational waveform. By simulating varying black hole populations in high-density globular clusters, we identify a population of highly eccentric binary black hole mergers that are characteristic of dynamical formation. Although these systems would circularize by the time they are visible in LIGO's frequency band, the future Laser Interferometer Space Antenna (LISA) is capable of distinguishing this population of eccentric mergers from the circular mergers expected of isolated field-formed binaries. As these dynamically formed binaries depend on the size of the underlying black hole population in globular clusters, we can utilize the dynamically formed merger rate to infer globular cluster black hole populations -- allowing us to reveal information about binary black hole birth environments.
In order to properly estimate the parameters of binary black holes from detected gravitational wave signals, such as their masses and spins, high-accuracy waveforms are a needed. The highest accuracy waveforms are those produced by numerical relativity simulations, which solve the full Einstein equations. Using the Spectral Einstein Code (SpEC), we expand the reach of numerical relativity to simulate binary black holes with nearly extremal spins, i.e., black holes with spins near the maximal value χ = 1. These waveforms are used to calibrate existing waveform approximants used in LIGO data analyses. This ensures that the systematic errors in these approximants are small enough that if highly-spinning systems are observed, the spins are recovered without bias. Although rapidly spinning binaries have remained elusive thus far, these waveforms ensure that the highest-spin systems can be detected in the quest to uncover the spin distribution of black holes.
The end state of a binary black hole merger is a newly born, single black hole that rings down like a struck bell, sending its last few ripples of gravitational waves out into the spacetime. Embedded in this 'ringdown' signal are a multitude of specific frequencies. Einstein's theory of general relativity precisely predicts the ringdown frequencies of a black hole with a given mass and spin. The statement that a black hole is entirely described by just these two parameters is known as the no-hair theorem. For black holes that obey the laws of general relativity (and consequently, the no-hair theorem), these frequencies serve as a fingerprint for the black hole. However, if the objects we observe are not Einstein's black holes, but instead something more exotic, the frequencies will not have this property and this would be a spectacular surprise. A minimum of two tones are required for this test, each with an associated frequency and damping time that depend only on the mass and spin. The conventional no-hair test relies on the so-called 'fundamental' tones of a black hole. A test relying on the fundamental modes is not expected to be feasible for another ~10-15 years, after detector sensitivity has improved significantly. However, by analyzing the ringdown of high-accuracy numerical relativity waveforms, we show that modes beyond the fundamental, known as 'overtones', are detectable in current detectors. The overtones are short-lived, but this is countered by the fact that they can initially be much stronger than the fundamental mode. By measuring two tones in the ringdown of GW150914 we perform a first test of the no-hair theorem. While the current constraints are rather loose, this first test serves as a proof of principle. This is just one example of the powerful tests that can be employed with overtones using present day detectors and the even more precise tests that can be accomplished with LISA in the future.</p
Essays on Social Learning and Networks
Get PDFThis thesis offers a contribution to the study of Social Learning and Networks. It studies information aggregation and its effect on individual's actions (Chapter 2, 3) and social network (Chapter 4).
Chapter 2, co-authored with Omer Tamuz and Wade Hann-Caruthers, studies how quickly does the public belief converge to its true value when agents are able to observe actions of their predecessors. In the classical herding literature, agents receive a private signal regarding a binary state of nature, and sequentially choose an action, after observing the actions of their predecessors. When the informativeness of private signals is unbounded, it is known that agents converge to the correct action and correct belief. We study how quickly convergence occurs, and show that it happens more slowly than it does when agents observe signals. However, we also show that the speed of learning from actions can be arbitrarily close to the speed of learning from signals. In particular, the expected time until the agents stop taking the wrong action can be either finite or infinite, depending on the private signal distribution. In the canonical case of Gaussian private signals, we calculate the speed of convergence precisely, and show explicitly that, in this case, learning from actions is significantly slower than learning from signals.
In Chapter 3, I investigate how social planning can reduce the inefficiencies of social learning, stemming from herding and informational cascades. A social planner is introduced to the classical sequential social learning model. She can tax or subsidize players' actions in order to maximize social welfare, a discounted sum of agents' utilities. We solve or accurately approximate the expected utility of the social planner and the optimal pricing strategy for various signal distributions. In equilibrium, it is optimal to increase the price for the better action, causing a reduction in current agent's utility, but also a net gain, due to the information this action reveals. The addition of the social planner significantly improves social welfare and the asymptotic speed of learning.
Chapter 4 analyzes how different types of social connections between people shape their social networks. There are two possible types of ties between individuals, strong and weak, that differ in maintenance costs and reliability. A network formation game is played in which agents choose the number of ties of each type to maximize their chances of hearing about a new job opportunity. We find that in equilibrium, people maintain both types of connections, which was not explained in previous theoretical models. Furthermore, in the socially optimal symmetric network, there are more strong ties than in the equilibrium one.</p
Enriching Majorana Zero Modes
Get PDFMy various projects in graduate school have centered around a common theme: harnessing relatively well-understood phases of matter and combining them to create exotic physics. They also involve Majoranas, or more accurately, defects that bind Majorana zero modes and are the centerpiece for topological quantum computation. We exploit and enrich this Majorana zero mode by employing topological superconductors, time crystals, and quantum dots and combining them together. Our first project involved joining Majorana nanowires and quantum dots to simulate the SYK model, a zero-dimensional strongly interacting phase with connections to black holes and holography. We follow by explaining how to combine spontaneous symmetry-breaking with topological superconductivity to recover parafermion physics in one dimension. We explain an exact mapping that relates fermions to parafermions, illustrating a deep connection between different one-dimensional phases of matter. We finally show that enhancing the topological superconductor with a time crystal, a phase of matter that spontaneously breaks time-translation symmetry, creates an anomalous zero mode that displays 4Tperiodicity in the Floquet drive. By combining these different phases in judicious ways we achieve exotic physics unattainable by the constituent parts. Our work thus illustrates profitable directions for harnessing Majorana zero modes to study the physics of exotic matter.</p
Expanding the Catalytic Repertoire of Hemeproteins as Carbene Transferases to Access Diverse Molecular Structures
Get PDFThe efficiency, selectivity, and sustainability benefits offered by enzymes are enticing chemists to consider biocatalytic transformations to complement or even supplant more traditional synthetic routes. Increasing demands for efficient and versatile synthetic methods combined with powerful new discovery and engineering tools have prompted innovations in biocatalysis, especially the development of new enzymes for precise transformations. The past decade has witnessed impressive expansion of the catalytic repertoire of enzymes to include new and useful transformations not known (or relevant) in the biological world. The continuing discovery and improvement of these new enzyme activities is opening a floodgate that connects the chemistry of the biological world to that invented by humans over the last 100 years.
This thesis describes a new set of enzymes, derived from a cytochrome P450 monooxygenase and a cytochrome c electron-transfer protein, which are able to function as carbene transferases to construct diverse molecular structures, including strained carbocycles and lactone derivatives. Chapter 1 illustrates different approaches researchers have utilized to explore and develop new catalytic machineries of diverse enzymes. These efforts have identified new genetically-encoded biocatalysts that can be tuned and diversified through directed evolution. Chapter 2 presents the discovery of P450 variants that catalyze the formation of highly strained carbocycles, bicyclobutanes and cyclopropenes, via carbene addition to carbon‒carbon triple bonds. The intrinsic strain energies of these small rigid carbocycles allow them to have broad applications in different fields, but also create challenges for their construction. Using a diazo substrate as the carbene precursor, the enzyme variants optimized by directed evolution could act on structurally diverse alkynes (aromatic or aliphatic, terminal or internal) with high efficiency and selectivity, providing an effective route to an array of chiral strained structures. The carbene transferase activity is then extended to the assembly of various lactone structures, a fundamental class of organic moieties with applications in fields varying from synthetic chemistry, to materials science, to medicinal chemistry. Chapter 3 details a strategy using lactone-based carbenes, for the transfer to different functionalities, enabling rapid access to a broad range of α-substituted and spiro-lactones with unprecedented efficiencies and selectivities. A different approach based on intramolecular carbene C–H insertion is outlined in Chapter 4, which allows for the synthesis of lactones in a higher order of structural diversity. Directed evolution of a P450 variant identified a lineage of potent variants, capable of assembling lactones in different sizes (5- to 7-membered) and also with sophisticated three-dimensional structures based on fused, spiro and bridged rings. Computational tools were employed to understand the reaction mechanisms and to explain some mutational effect. In sum, the thesis work lays out how protein engineering integrated with chemical rationalization enables the expansion of the chemical space accessible to native hemeproteins, especially in building diverse molecular structures.</p
Quantum Mechanical Vistas on the Road to Quantum Gravity
Get PDFIn this thesis, we lay out the goal, and a broad outline, for a program that takes quantum mechanics in its minimal form to be the fundamental ontology of the universe. Everything else, including features like space-time, matter and gravity associated with classical reality, are emergent from these minimal quantum elements. We argue that the Hilbert space of quantum gravity is locally finite-dimensional, in sharp contrast to that of conventional field theory, which could have observable consequences for gravity. We also treat time and space on an equal footing in Hilbert space in a reparametrization invariant setting and show how symmetry transformations, both global and local, can be treated as unitary basis changes.
Motivated by the finite-dimensional context, we use Generalized Pauli Operators as finite-dimensional conjugate variables and define a purely Hilbert space notion of locality based on the spread induced by conjugate operators which we call "Operator Collimation." We study deviations in the spectrum of physical theories, particularly the quantum harmonic oscillator, induced by finite-dimensional effects, and show that by including a black hole-based bound in a lattice field theory, the quantum contribution to the vacuum energy can be suppressed by multiple orders of magnitude.
We then show how one can recover subsystem structure in Hilbert space which exhibits emergent quasi-classical dynamics. We explicitly connect classical features (such as pointer states of the system being relatively robust to entanglement production under environmental monitoring and the existence of approximately classical trajectories) with features of the Hamiltonian. We develop an in-principle algorithm based on extremization of an entropic quantity that can sift through different factorizations of Hilbert space to pick out the one with manifest classical dynamics. This discussion is then extended to include direct sum decompositions and their compatibility with Hamiltonian evolution.
Following this, we study quantum coarse-graining and state-reduction maps in a broad context. In addition to developing a first-principle quantum coarse-graining algorithm based on principle component analysis, we construct more general state-reduction maps specified by a restricted set of observables which do not span the full algebra (as could be the case of limited access in a laboratory or in various situations in quantum gravity). We also present a general, not inherently numeric, algorithm for finding irreducible representations of matrix algebras.
Throughout the thesis, we discuss implications of our work in the broader goal of understanding quantum gravity from minimal elements in quantum mechanics.</p
Structure, Function, and Application of Bacterial ABC Transporters
Get PDFThe ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single particle cryo-EM in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione binding site and the associated substrate binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provides an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide binding domains in the inward-facing conformation.
The ATPase kinetic data was fit to a non-essential activator model with expansion to two substrate binding sites. While the structural data suggests that MgATP and GSSG bind to distinct states, outward- and inward-facing conformations, respectively, and hence might be expected to exhibit negative cooperativity, the kinetic data support a more complex interplay and also the importance of lipid molecule presence. How GSSG binding stimulates ATPase activity remains an open question and highlights the importance of the still elusive ternary complex with both MgATP and GSSG bound to NaAtm1.
Besides the structural and functional characterizations of the ABC exporter, NaAtm1, we additionally determined crystal structures of the repurposed periplasmic binding protein (PBP) from the ABC importer system. These PBPs are designed with circularly permutated GFP to act as biosensors to sense the concentrations of smoking cessation drugs and neurotransmitters under cellular conditions. The crystal structures determined for the nicotine and acetylcholine biosensors not only revealed the key residues in ligand binding, but also demonstrated similar ligand induced conformational changes as seen in other PBPs by following the Venus-flytrap mechanism.</p
Neural Construction of Conscious Perception
Get PDFOut of a myriad of sensory stimulations, our brain constructs a unified, self-consistent reality that we consciously experience. Little is known about how or where in the brain’s processing stream of physical input a conscious percept emerges into awareness. A remarkable property of conscious perception is that even though external input is often ambiguous, the perceptual interpretation of the world that our brain generates is consistent across multiple layers of representation, e.g., figure-ground segmentation and object identity. We thus set out to study how the interaction between different nodes in the brain generates and propagates new conscious percepts. Since the code of object identity is already well-understood, in particular for faces as reviewed in this thesis, we decided to get a handle on segmentation signals first. It turned out that consistent segmentation signals are hard to find, however, we found functionally defined modules in the brain that contained consistent cells from which figure-ground signals can be decoded. We next investigated whether face cells in object recognition areas actually encode the conscious percept of a face or are just passive filters of visual input. To distill conscious perception from other cognitive processes, such as decision making, introspection, and reporting of the percept, which often accompany new conscious percepts, we developed a no-report binocular rivalry paradigm that relies on an active fixation task rather than report, and therefore eliminates these confounding factors. We found that face patches in inferotemporal cortex indeed encode the conscious percept of a face. Using novel high-yield electrodes, we were able to decode what the animal was consciously perceiving at a given time. Preliminary and future experiments of population recordings from multiple nodes of the cortical hierarchy simultaneously promise to go beyond correlates of consciousness and reveal the mechanisms of how and where conscious percepts are constructed.</p