RNA ligase ribozymes with a small catalytic core.

Catalytic RNAs, or ribozymes, catalyze diverse chemical reactions that could have sustained primordial life in the hypothetical RNA world. Many natural ribozymes and laboratory evolved ribozymes exhibit efficient catalysis mediated by elaborate catalytic cores within complex tertiary structures. However, such complex RNA structures and sequences are unlikely to have emerged by chance during the earliest phase of chemical evolution. Here, we explored simple and small ribozyme motifs capable of ligating two RNA fragments in a template-directed fashion (ligase ribozymes). One-round selection of small ligase ribozymes followed by deep sequencing revealed a ligase ribozyme motif comprising a three-nucleotide loop opposite to the ligation junction. The observed ligation was magnesium(II) dependent and appears to form a 2'-5' phosphodiester linkage. The fact that such a small RNA motif can function as a catalyst supports a scenario in which RNA or other primordial nucleic acids played a central role in chemical evolution of life.

Chemical control of phase separation in DNA solutions.

We designed a series of DNA sequences comprising a trinucleotide repeat segment and a small molecule-binding aptamer. Optimization of the DNA sequences and reaction conditions enabled chemical control of phase separation of DNA condensates. Our results demonstrate a new strategy to regulate biomolecular phase transition.

Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals.

Synthetic riboswitches that can regulate gene expression by a small molecule recognized by an RNA aptamer in mammalian cells have various potential applications in biotechnology and medicine. However, the variety of small molecules and their cognate aptamers that have been demonstrated to function in mammalian cells is limited. The currently available aptamer-ligand pairs also require high small molecule concentrations to enable gene regulation, making them less desirable for industrial and biomedical applications. We conducted in vitro selection of RNA aptamers against a small molecule ASP7967 whose structure is closely related to ASP2905, a known inhibitor of potassium voltage-gated channel sub-family H member 3 (KCNH3). One of the aptamers selected (AC17-4) was found to be functional in HEK293 cells, and it was used to design aptazyme-based riboswitches that can activate gene expression (>10-fold) in the presence of ASP2905 or ASP7967 at as low as 5 µM in the culture medium. An aptazyme-based riboswitch was successfully used to regulate human erythropoietin expression in mice injected with an adeno-associated virus (AAV8) vector using orally administered ASP7967. Furthermore, by combining aptazyme-based and exon-skipping riboswitch mechanisms, an ON/OFF ratio approaching 300 was achieved with a low basal expression level in cultured cells.

Experimental exploration of a ribozyme neutral network using evolutionary algorithm and deep learning.

A neutral network connects all genotypes with equivalent phenotypes in a fitness landscape and plays an important role in the mutational robustness and evolvability of biomolecules. In contrast to earlier theoretical works, evidence of large neutral networks has been lacking in recent experimental studies of fitness landscapes. This suggests that evolution could be constrained globally. Here, we demonstrate that a deep learning-guided evolutionary algorithm can efficiently identify neutral genotypes within the sequence space of an RNA ligase ribozyme. Furthermore, we measure the activities of all 216 variants connecting two active ribozymes that differ by 16 mutations and analyze mutational interactions (epistasis) up to the 16th order. We discover an extensive network of neutral paths linking the two genotypes and reveal that these paths might be predicted using only information from lower-order interactions. Our experimental evaluation of over 120,000 ribozyme sequences provides important empirical evidence that neutral networks can increase the accessibility and predictability of the fitness landscape.

Analysis of the Sequence Preference of Saporin by Deep Sequencing.

Ribosome-inactivating proteins (RIPs) are RNA:adenosine glycosidases that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin loop (SRL) in 28S rRNA. The GAGA sequence at the top of the SRL or at the top of a hairpin loop is assumed to be their target motif. Saporin is a RIP widely used to develop immunotoxins for research and medical applications, but its sequence specificity has not been investigated. Here, we combine the conventional aniline cleavage assay for depurinated nucleic acids with high-throughput sequencing to study sequence-specific depurination of oligonucleotides caused by saporin. Our data reveal the sequence preference of saporin for different substrates and show that the GAGA motif is not efficiently targeted by this protein, neither in RNA nor in DNA. Instead, a preference of saporin for certain hairpin DNAs was observed. The observed sequence-specific activity of saporin may be relevant to antiviral or apoptosis-inducing effects of RIPs. The developed method could also be useful for studying the sequence specificity of depurination by other RIPs or enzymes.

Speed variations of bacterial replisomes.

Replisomes are multi-protein complexes that replicate genomes with remarkable speed and accuracy. Despite their importance, their dynamics is poorly characterized, especially in vivo. In this paper, we present an approach to infer the replisome dynamics from the DNA abundance distribution measured in a growing bacterial population. Our method is sensitive enough to detect subtle variations of the replisome speed along the genome. As an application, we experimentally measured the DNA abundance distribution in Escherichia coli populations growing at different temperatures using deep sequencing. We find that the average replisome speed increases nearly fivefold between 17 °C and 37 °C. Further, we observe wave-like variations of the replisome speed along the genome. These variations correlate with previously observed variations of the mutation rate, suggesting a common dynamical origin. Our approach has the potential to elucidate replication dynamics in E. coli mutants and in other bacterial species.

Identification of an ERN1 target site within EGFP mRNA.

EGFP (enhanced green fluorescent protein) is one of the most common tools used in life sciences, including research focusing on proteostasis. Here we report that ERN1 (endoplasmic reticulum to nucleus signaling 1), which is upregulated by UPR (unfolded protein response), targets an RNA hairpin loop motif in EGFP mRNA. A silent mutation introduced into EGFP mRNA abolished the ERN1-dependent mRNA decay. Therefore, experiments that employ EGFP as a reporter gene in studies that involve upregulation of the UPR pathway should be interpreted carefully, and a mutant devoid of the ERN1 target motif may be more suitable for such studies.

High-throughput screening of cell-free riboswitches by fluorescence-activated droplet sorting.

Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets.

Programmable Macroscopic Self-Assembly of DNA-Decorated Hydrogels.

The precise and predictable formation of double-helical structures from complementary DNA sequences has made DNA an extremely versatile tool for programming self-assembled structures from the nanometer to micrometer scale. While a number of supramolecular interactions have been shown to drive self-assembly of macroscopic building blocks of the millimeter scale, DNA-driven self-assembly of macroscopic objects has not been well-established. In this work, we developed a postpolymerization coupling strategy to conjugate short DNA sequences to polyacrylamide-based hydrogel blocks. We observed sequence-specific self-assembly of DNA-decorated hydrogels with 1-2 mm edges in aqueous solution. Furthermore, selective disassembly of hydrogels upon addition of a DNA strand was demonstrated by exploiting a strand displacement reaction. These results lay the foundation for adaptation of various DNA functions to macroscopic self-assembly, for example, molecular recognition, molecular computation, and chemical catalysis.

Directed evolution of orthogonal RNA-RBP pairs through library-vs-library in vitro selection.

RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA-RBP pairs from natural components remains challenging while such synthetic RNA-RBP pairs could significantly expand the RNA-RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 1012 unique RNA sequences and 4.0 × 108 unique phage-displayed L7Ae-scaffold (LS) proteins, we selected RNA-RBP complexes through a two-step affinity purification process. After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA-RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.

Novel RNA Viral Vectors for Chemically Regulated Gene Expression in Embryonic Stem Cells.

RNA viral vectors that replicate without DNA intermediates are attractive platforms for manipulation of cells for biomedical and veterinary applications because they have minimal risk of chromosomal integration. Vesicular stomatitis virus (VSV) vectors are among the most well-studied RNA viral vectors due to their low pathogenicity to humans and ability to express transgenes at high levels for weeks to months. However, their applications have been mostly limited to oncolytic and vaccine vectors due to their cytopathogenicity. We discovered two mutations in the VSV vector that synergistically confer improved stability in mouse embryonic stem cells (ESCs) with markedly lower cytopathic effects. We also demonstrated chemical regulation of transgene expression through embedded riboswitches. The ESCs infected with the mutant vector were shown to maintain pluripotency. This new vector sets the stage for precise regulation of gene expression in ESCs to produce a variety of differentiated cells without chromosomal alteration.

Cell-free riboswitches.

The emerging community of cell-free synthetic biology aspires to build complex biochemical and genetic systems with functions that mimic or even exceed those in living cells. To achieve such functions, cell-free systems must be able to sense and respond to the complex chemical signals within and outside the system. Cell-free riboswitches can detect chemical signals via RNA-ligand interaction and respond by regulating protein synthesis in cell-free protein synthesis systems. In this article, we review synthetic cell-free riboswitches that function in both prokaryotic and eukaryotic cell-free systems reported to date to provide a current perspective on the state of cell-free riboswitch technologies and their limitations.

Circularly-Permuted Pistol Ribozyme: A Synthetic Ribozyme Scaffold for Mammalian Riboswitches.

A small molecule-responsive self-cleaving ribozyme (aptazyme) embedded in the untranslated region of an mRNA functions as a riboswitch that allows chemical regulation of gene expression in mammalian cells. Aptazymes are engineered by fusing a self-cleaving ribozyme with an RNA aptamer that recognizes a small molecule so that the ribozyme is either activated or inhibited in the presence of the small molecule. However, the variety of aptamers, ribozymes, and aptazyme design strategies suitable for mammalian riboswitch applications is still limited. This work focuses on a new ribozyme scaffold for engineering aptazymes and riboswitches that function in mammalian cells. We investigated circularly permuted variants of the pistol ribozyme class (CPP) as a synthetic ribozyme scaffold for mammalian riboswitch applications. Through semirational design and high-throughput screening, we designed guanine and tetracycline activated riboswitches based on three distinct aptazyme architectures, resulting in riboswitches with ON/OFF ratios as high as 8.6. Our work adds CPP to the limited ribozyme scaffold toolbox for mammalian synthetic biology applications and highlights the opportunities in exploring ribozymes beyond natural motifs.

Aptazyme-Based Riboswitches and Logic Gates in Mammalian Cells.

This chapter describes a screening strategy to engineer synthetic riboswitches that can chemically regulate gene expression in mammalian cells. Riboswitch libraries are constructed by randomizing the key nucleotides that couple the molecular recognition function of an aptamer with the self-cleavage activity of a ribozyme. The allosteric ribozyme (aptazyme) candidates are cloned in the 3' untranslated region (UTR) of a reporter gene mRNA. The plasmid-encoded riboswitch candidates are transfected into a mammalian cell line to screen for the desired riboswitch function. Furthermore, multiple aptazymes can be cloned into the 3' UTR of a desired gene to obtain a logic gate response to multiple chemical signals. This screening strategy complements other methods to engineer robust mammalian riboswitches to control gene expression.

Ribozyme Mutagenic Evolution: Mechanisms of Survival.

Primeval populations replicating at high error rates required a mechanism to overcome the accumulation of mutations and information deterioration. Known strategies to overcome mutation pressures include RNA processivity, epistasis, selection, and quasispecies. We investigated the mechanism by which small molecular ribozyme populations can survive under high error rates by propagating several lineages under different mutagen concentrations. We found that every population that evolved without mutagen went extinct, while those subjected to mutagenic evolution survived. To understand how they survived, we characterized the evolved genotypic diversity, the formation of genotype-genotype interaction networks, the fitness of the most common mutants for each enzymatic step, and changes in population size along the course of evolution. We found that the elevated mutation rate was necessary for the populations to survive in the novel environment, in which all the steps of the metabolism worked to promote the survival of even less catalytically efficient ligases. Besides, an increase in population size and the mutational coupling of genotypes in close-knit networks, which helped maintain or recover lost genotypes making their disappearance transient, prevented Muller's ratchet and extinction.

High-Throughput Analysis and Engineering of Ribozymes and Deoxyribozymes by Sequencing.

Ribozymes and deoxyribozymes are catalytic RNA and DNA, respectively, that catalyze chemical reactions such as self-cleavage or ligation reactions. While some ribozymes are found in nature, a larger variety of ribozymes and deoxyribozymes have been discovered by in vitro selection from random sequences. These catalytic nucleic acids, especially ribozymes, are of fundamental interest because they are crucial for the RNA world hypothesis, which suggests that RNA played a central role in both the propagation of genetic information and catalyzing metabolic reactions in primordial life prior to the emergence of proteins and DNA. On the practical side, catalytic nucleic acids have been extensively engineered for various applications, such as biosensors and genetic devices for synthetic biology. Therefore, it is important to gain a deeper understanding of the sequence-function relationships of ribozymes and deoxyribozymes.Mutational analysis, or measurements of activities of catalytic nucleic acid mutants, is one of the most fundamental approaches for that purpose. Mutations that abolish, reduce, retain, or even increase activity provide useful information about nucleic acid catalysts for engineering and other purposes. However, methods for mutational analysis of ribozymes and deoxyribozymes have not evolved much for decades, requiring tedious and low-throughput assays (e.g., gel electrophoresis) of individually prepared mutants. This has prevented researchers from performing quantitative mutational analysis of ribozymes and deoxyribozymes on a large scale.To address this limitation, we developed a massively parallel ribozyme and deoxyribozyme assay strategy that allows >104 assays using high-throughput sequencing (HTS). We used HTS to literally count the number of cleaved (or ligated) and uncleaved (or unligated) ribozyme (or deoxyribozyme) sequences and calculated the activities of each mutant in a reaction mixture. This simple yet powerful strategy was applied to analyze the mutational effects of various natural and synthetic ribozymes and deoxyribozymes at scales impossible for conventional mutational analysis. These large-scale sequence-function data sets were used to better understand the functional consequences of mutations and to engineer ribozymes for practical applications. Furthermore, these newly available data are motivating researchers to employ more rigorous computational methods to extract additional insights such as structural information and nonlinear effects of multiple mutations. The new HTS-based assay strategy is distinct from and complementary to a related strategy that uses HTS to analyze ribozyme and deoxyribozyme populations subjected to in vitro selection. Postselection sequencing can cover a larger sequence space, although it does not directly quantify the activities of ribozyme and deoxyribozyme mutants. With further advances in DNA sequencing technologies and computational methods, there should be more opportunities to harness the power of HTS to deepen our understanding of catalytic nucleic acids and enhance our ability to engineer them for even more applications.

Design of Mammalian ON-Riboswitches Based on Tandemly Fused Aptamer and Ribozyme.

Self-cleaving ribozymes engineered to be activated or inhibited by a small molecule binding to an RNA aptamer inserted within a ribozyme (aptazymes) have proven to be useful for controlling gene expression in living cells. In mammalian cells, an aptazyme embedded in the 5' or 3' untranslated region of an mRNA functions as a synthetic riboswitch to chemically regulate gene expression. However, the variety of aptazyme architectures and the ribozyme scaffolds that have been used for mammalian riboswitches has been limited. In particular, fewer synthetic riboswitches that activate gene expression in response to a small molecule (ON-switches) in mammalian cells have been reported compared to OFF-switches. In this work, we developed mammalian riboswitches that function as guanine-activated ON-switches based on a novel aptazyme architecture in which an aptamer and a ribozyme are fused in tandem. The riboswitch performance was optimized by fine-tuning the stability of a critical stem that controls the ribozyme structure and function, yielding switches with ON/OFF ratios greater than 6.0. Our new aptazyme architecture expands the RNA device toolbox for controlling gene expression in mammalian cells.