Improving the Ribozyme Toolbox: From Structure-Function Insights to Synthetic Biology Applications

Self-cleaving ribozymes are a naturally occurring class of catalytically active RNA molecules which cleave their own phosphate backbone. In nature, self-cleaving ribozymes are best known for their role in processing concatamers of viral genomes into monomers during viral replication in some RNA viruses, but to a lesser degree have also been implicated in mRNA regulation and processing in bacteria and eukaryotes. In addition to their biological relevance, these RNA enzymes have been harnessed as important biomolecular tools with a variety of applications in fields such as bioengineering. Self-cleaving ribozymes are relatively small and easy to generate in the lab using common molecular biology approaches, and have therefore been accessible and well exploited model systems used to interrogate RNA sequence-structure-function relationships. Furthermore, self-cleaving ribozymes are also being implemented as parts in the development of various biomolecular tools such as biosensors and gene regulatory elements. While much progress has been made in these areas, there are still challenges associated with the performance and implementation of such tools. The work contained in this dissertation aims to address several of these challenges and improve the ribozyme toolbox in several diverse areas. Chapter one provides an introduction to pertinent background information for this dissertation. Chapter two aims to improve the ribozyme toolbox by providing and analyzing new high-throughput sequence-structure-function data sets on five different self-cleaving ribozymes, and identifying how trends in epistasis relate to distinct structural elements. Chapter three uses such high-throughput data to train machine learning models that accurately predict the historically difficult to predict functional effects of higher order mutations in functional RNA’s. Finally, in chapter four, I developed a biologically relevant platform to study the real time performance and kinetics of self-cleaving ribozyme-based gene regulatory elements directly at the site of transcription in mammalian cells

Empirical Investigations OF RNA Fitness Landscapes: Harnessing the Power of High-Throughput Sequencing and Evolutionary Simulations

Fitness landscapes or adaptive landscapes represent the mapping of genotype (sequence) to phenotype (function or fitness). Originally proposed as a metaphor to envision evolutionary processes and mutational interactions, the fitness landscape has recently transitioned from theoretical to empirical. This is due in part to advances in DNA synthesis and high-throughput sequencing. This allows for the construction and analysis of empirical fitness landscapes that encompass thousands of genotypes. These landscapes provide tractable insight into mutational pathways, the predictability of evolution or even the evolution of life. RNA enzymes (ribozymes) are an attractive model system for the construction of empirical fitness landscapes. Ribozymes function as both a genotype (primary RNA sequence) and a phenotype (catalytic function). To construct and characterize empirical RNA fitness landscapes, two high-throughput functional assays (self-cleavage and self-ligation), including a technique to improve data recovery from high-throughput sequencing using phased nucleotide inserts (Appendix A), were developed and implemented. Following fitness landscape construction, a stochastic evolutionary model was developed and employed based on the Wright-Fisher model. This model follows the principles of Darwinian evolution and allows a population to explore the fitness landscape by means of mutation and selection. These newly developed tools allowed for a novel approach to important evolutionary questions. Chapter 1 explored the evolution of innovation at the intersection of two ribozyme functions: self-cleavage and self-ligation. Evolutionary innovations are qualitatively novel traits that emerge through evolution. Theories have suggested that innovations can occur where two genotype networks are in close proximity. However, only isolated examples of intersections have been investigated. The fitness landscape between the two ribozyme functions was explored by determining the ability of numerous neighboring RNA sequences to catalyze two different chemical reactions. This revealed that there was extensive functional overlap, and over half the genotypes can catalyze both functions to some extent. Data-driven evolutionary simulations found that these numerous points of intersection facilitated the discovery of a new function, yet the rate of optimization depended upon the starting location in the genotype network. This study constructed a fitness landscape where genotype networks intersect and uncovered the implications for evolutionary innovations. Chapter 2 determined the effect of higher sequence space complexity and dimensionality on evolutionary adaptation in RNA fitness landscapes. The complexity and dimensionality of landscapes scale with the length of the RNA molecule. For this study, complexity was defined as the size of the genotype space and dimensionality as the number of edges connecting each genotype (node) to other genotypes that differ by a single mutation. Low-dimensional ‘direct’ landscapes consisting of only two possible nucleotides at various positions were compared to higher-dimensional ‘indirect’ landscapes that had all four nucleotides at the same positions. Indirect pathways contributed to the ruggedness and navigability of landscapes. Increased dimensionality in RNA fitness landscapes had the potential to circumvent fitness valleys, however indirect pathways also harbored stasis genotypes isolated by reciprocal sign epistasis. Chapter 3 applied ancestral sequence resurrection and fitness landscape construction to naturally evolved ribozymes. The CPEB3 ribozyme is highly conserved in mammals and has been linked to episodic memory. By predicting, ‘resurrecting’ and functionally characterizing ancient gene sequences, hypotheses about gene function or selection can be empirically tested in an evolutionary context. Using the extant ribozyme sequences found in a range of mammalian species as a basis for inference of ancestral sequences, a phylogenetic fitness landscape was experimentally resurrected and reconstructed. A single high-activity ancestral sequence was found to be highly conserved and purifying selection is expected to have reduced the accumulation of mutations through geologic time. Many of the extant mammalian ribozyme sequences had high ribozyme activity, however a few had relatively low activity. Yet, given the local fitness landscape, a selective pressure for functional ribozyme sequences was seen. A single nucleotide polymorphism (SNP) found in humans, reduced co-transcriptional ribozyme activity in vitro and might alter our understanding of the CPEB3 ribozyme’s biological function. Chapter 4 analyzed epistatic interactions in four published RNA fitness landscapes generated from high-throughput analyses. Two of the landscapes were assessed in vivo and two were assessed in vitro. Epistasis occurs when the effects of some mutations are dependent on the presence or absence of other mutations. The data allowed for an analysis of the distribution of fitness effects of individual mutations as well as combinations of two or more mutations. Two different approaches to measuring epistasis in the data both revealed a predominance of negative epistasis, such that higher combinations of two or more mutations are typically lower in fitness than expected from the effect of each individual mutation. This finding differed from studies using computationally predicted RNA but is similar to mutational experiments in protein enzymes. The work presented here represents a significant contribution to our ability to construct and empirically characterize RNA fitness landscapes. The development of two high-throughput ribozyme assays opens the door for further empirical landscape construction. The implementation of data-driven stochastic evolutionary modeling allows for a clearer evolutionary characterization of the landscape. Understanding the connection between genotype and phenotype in RNA systems is important for designing RNA functions, improving in vitro selections and understanding the origins and evolution of new RNA functions (innovations). Applying these advances yielded valuable information about evolutionary innovations, the effects of higher dimensionality, evolution of extant ribozymes and the prevalence of epistasis in RNA fitness landscapes. Construction and analysis of empirical RNA fitness landscapes provides tractable insight into evolutionary processes, mutational pathways and the predictability of evolution

Selection of an aptamer and development of a genetic device to control mRNA stability in response to light

Artificial autocatalytic RNA molecules, so-called aptazymes, are utilized as genetic devices to study and influence biological processes. These genetic devices consist of the autocatalytic RNA part, the ribozyme, and a ligand-sensing part, called aptamer. Most of these devices use small molecule ligands as a trigger to regulate the cleavage activity of the aptazyme. However, the utility of small molecules has some limitations such as potential side effects, lack of reversibility as well as limited spatial and temporal control. In contrast, visible light as a trigger has the advantage of being reversible, non-harmful, and spatially and temporally controllable. Ribozymes per se, however, have no intrinsic ability to sense light. But in the field of optogenetics, proteins with LOV domains act as light-sensitive sensors. Herein, optogenetics was combined with aptamer technology to develop a genetic device capable of regulating mRNA stability in response to light. In a rational design approach, an aptamer that can bind the LOV protein PAL was fused to the hammerhead ribozyme. The aptazyme exhibited light-dependent cleavage activity, which could be modulated by varying the stem length of the introduced aptamer. This concept created aptazymes that exhibited decreased or increased cleavage activity in the presence of light. This discovery paves the way for the utilization of this system in the field of synthetic biology or basic research.Künstliche autokatalytische RNA-Moleküle, so genannte Aptazyme, werden als genetische Werkzeuge zur Untersuchung und Beeinflussung biologischer Prozesse eingesetzt. Diese genetischen Werkzeuge bestehen aus dem autokatalytischen RNA-Teil, dem Ribozym, und einem ligandensensitiven Teil, dem Aptamer. Die meisten dieser Werkzeuge verwenden niedermolekulare Liganden als Auslöser, um die Spaltungsaktivität des Aptazyms zu regulieren. Die Verwendung niedermolekularer Liganden hat jedoch einige Limitationen, beispielsweise potenzielle Nebenwirkungen, mangelnde Reversibilität sowie begrenzte räumliche und zeitliche Kontrolle. Im Gegensatz dazu hat sichtbares Licht als Auslöser den Vorteil, dass es reversibel, nicht schädlich und räumlich und zeitlich kontrollierbar ist. Ribozyme als solche besitzen nicht die intrinsische Fähigkeit, Licht zu perzipieren. Im Bereich der Optogenetik fungieren Proteine mit LOV-Domänen jedoch als lichtempfindliche Sensoren. In diesem Projekt wurde die Optogenetik mit der Aptamer-Technologie kombiniert, um ein genetisches Werkzeug zu entwickeln, das die mRNA-Stabilität als Reaktion auf Licht regulieren kann. In einem rationalen Designansatz wurde ein Aptamer, das das LOV-Protein PAL binden kann, mit dem Hammerhead-Ribozym fusioniert. Das Aptazym zeigte eine lichtabhängige Spaltungsaktivität, die durch Variation der Stammlänge des angebundenen Aptamers moduliert werden konnte. Mit diesem Konzept wurden Aptazyme geschaffen, die in Gegenwart von Licht eine verringerte oder erhöhte Spaltaktivität aufweisen. Diese Entdeckung ebnet den Weg für die Nutzung dieses Systems im Bereich der synthetischen Biologie oder für die Grundlagenforschung

Reversible Gene Regulation in Mammalian Cells Using Riboswitch-Engineered Vesicular Stomatitis Virus Vector

Synthetic riboswitches based on small molecule-responsive self-cleaving ribozymes (aptazymes) embedded in the untranslated regions (UTRs) allow chemical control of gene expression in mammalian cells. In this work, we used a guanine-responsive aptazyme to control transgene expression from a replication-incompetent vesicular stomatitis virus (VSV) vector. VSV is a nonsegmented, negative-sense, cytoplasmic RNA virus that replicates without DNA intermediates, and its applications for vaccines and oncolytic viral therapy are being explored. By inserting the guanine-activated ribozyme in the 3′ UTRs of viral genes and transgenes, GFP expression from the VSV vector in mammalian cells was repressed by as much as 26.8-fold in the presence of guanine. Furthermore, we demonstrated reversible regulation of a transgene (secreted NanoLuc) by adding and withdrawing guanine from the medium over the course of 12 days. In summary, our riboswitch-controlled VSV vector allows robust, long-term, and reversible regulation of gene expression in mammalian cells without the risk of undesirable genomic integration

Selection platforms for directed evolution in synthetic biology

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules – gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function – be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code

Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV Ribozyme

The HDV ribozyme’s folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U23 and G28 nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U23 and G28 can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction

Synthetic biology: advancing biological frontiers by building synthetic systems

Advances in synthetic biology are contributing to diverse research areas, from basic biology to biomanufacturing and disease therapy. We discuss the theoretical foundation, applications, and potential of this emerging field

Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome

Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNAval promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes

Rapid Evolution of Manifold CRISPR Systems for Plant Genome Editing

Advanced CRISPR-Cas9 based technologies first validated in mammalian cell systems are quickly being adapted for use in plants. These new technologies increase CRISPR-Cas9’s utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. Here, we review the current state of advanced CRISPR-Cas9 and Cpf1 capabilities in plants and cover the rapid evolution of these tools from first generation inducers of double strand breaks for basic genetic manipulations to second and third generation multiplexed systems with myriad functionalities, capabilities, and specialized applications. We offer perspective on how to utilize these tools for currently untested research endeavors and analyze strengths and weaknesses of novel CRISPR systems in plants. Advanced CRISPR functionalities and delivery options demonstrated in plants are primarily reviewed but new technologies just coming to the forefront of CRISPR development, or those on the horizon, are briefly discussed. Topics covered are focused on the expansion of expression and delivery capabilities for CRISPR-Cas9 components and broadening targeting range through orthogonal Cas9 and Cpf1 proteins