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.

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.

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.

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.

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.

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.

Systematic minimization of RNA ligase ribozyme through large-scale design-synthesis-sequence cycles.

Template-directed RNA ligation catalyzed by an RNA enzyme (ribozyme) is a plausible and important reaction that could have been involved in transferring genetic information during prebiotic evolution. Laboratory evolution experiments have yielded several classes of ligase ribozymes, but their minimal sequence requirements remain largely unexplored. Because selection experiments strongly favor highly active sequences, less active but smaller catalytic motifs may have been overlooked in these experiments. We used large-scale DNA synthesis and high-throughput ribozyme assay enabled by deep sequencing to systematically minimize a previously laboratory-evolved ligase ribozyme. After designing and evaluating >10 000 sequences, we identified catalytic cores as small as 18 contiguous bases that catalyze template-directed regiospecific RNA ligation. The fact that such a short sequence can catalyze this critical reaction suggests that similarly simple or even simpler motifs may populate the RNA sequence space which could have been accessible to the prebiotic ribozymes.

Self-powered RNA nanomachine driven by metastable structure.

Many non-coding and regulatory RNA elements have evolved to exploit transient or metastable structures that emerge during transcription to control complex folding pathways or to encode dynamic functions. However, efforts to engineer synthetic RNA devices have mostly focused on the thermodynamically stable structures. Consequently, significant challenges and opportunities exist in engineering functional RNAs that explicitly take advantage of cotranscriptionally generated transient or metastable structures. In this work, we designed a short RNA sequence that adopts a robust metastable structure when transcribed by an RNA polymerase. Although the metastable structure persists for hours at low temperature, it refolds almost completely into the thermodynamically stable structure upon heat denaturation followed by cooling. The synthetic RNA was also equipped with the Broccoli aptamer so that it can bind its ligand and become fluorescent only in the thermodynamically stable structure. We further demonstrated that the relaxation to the thermodynamically stable and fluorescent structure can be catalyzed by a short trigger RNA in a sequence-specific manner. Finally, the RNA architecture was redesigned to sense and respond to microRNA sequences. In summary, we designed RNA nanomachines that can detect an RNA sequence, amplify signal and produce an optical output, all encoded in a single RNA transcript, self-powered by a metastable structure.

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.

Applications of high-throughput sequencing to analyze and engineer ribozymes.

A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such as regulation of gene expression and viral replication, but biological roles of many ribozymes still remain unknown. Ribozymes have also inspired researchers to engineer synthetic ribozymes that function as sensors or gene switches. To gain deeper understanding of the sequence-function relationship of ribozymes and to efficiently engineer synthetic ribozymes, a large number of ribozyme variants need to be examined which was limited to hundreds of sequences by Sanger sequencing. The advent of high-throughput sequencing technologies, however, has allowed us to sequence millions of ribozyme sequences at low cost. This review focuses on the recent applications of high-throughput sequencing to both characterize and engineer ribozymes, to highlight how the large-scale sequence data can advance ribozyme research and engineering.

Programmable Artificial Cells Using Histamine-Responsive Synthetic Riboswitch.

Artificial cells that encapsulate DNA-programmable protein expression machinery are emerging as an attractive platform for studying fundamental cellular properties and applications in synthetic biology. However, interfacing these artificial cells with the complex and dynamic chemical environment remains a major and urgent challenge. We demonstrate that the repertoire of molecules that artificial cells respond to can be expanded by synthetic RNA-based gene switches, or riboswitches. We isolated an RNA aptamer that binds histamine with high affinity and specificity and used it to design robust riboswitches that activate protein expression in the presence of histamine. Finally, the riboswitches were incorporated in artificial cells to achieve controlled release of an encapsulated small molecule and to implement a self-destructive kill-switch. Synthetic riboswitches should serve as modular and versatile interfaces to link artificial cell phenotypes with the complex chemical environment.

High-throughput assay and engineering of self-cleaving ribozymes by sequencing.

Self-cleaving ribozymes are found in all domains of life and are believed to play important roles in biology. Additionally, self-cleaving ribozymes have been the subject of extensive engineering efforts for applications in synthetic biology. These studies often involve laborious assays of multiple individual variants that are either designed rationally or discovered through selection or screening. However, these assays provide only a limited view of the large sequence space relevant to the ribozyme function. Here, we report a strategy that allows quantitative characterization of greater than 1000 ribozyme variants in a single experiment. We generated a library of predefined ribozyme variants that were converted to DNA and analyzed by high-throughput sequencing. By counting the number of cleaved and uncleaved reads of every variant in the library, we obtained a complete activity profile of the ribozyme pool which was used to both analyze and engineer allosteric ribozymes.

High-Throughput Mutational Analysis of a Twister Ribozyme.

Recent discoveries of new classes of self-cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self-cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations.

Switchable and orthogonal gene expression control inside artificial cells by synthetic riboswitches.

Here we report two novel synthetic riboswitches that respond to ASP2905 and theophylline and function in reconstituted cell-free protein synthesis (CFPS) system. We encapsulated the CFPS system as well as DNA-templated encoding reporter genes regulated by these orthogonal riboswitches inside liposomes, and achieved switchable and orthogonal control over gene expression by external stimulation with the cognate ligands.

Optimization of Exon-Skipping Riboswitches and Their Applications to Control Mammalian Cell Fate.

Mammalian riboswitches that can regulate transgene expression via RNA-small molecule interaction have promising applications in medicine and biotechnology, as they involve no protein factors that can induce immunogenic reactions and are not dependent on specially engineered promoters. However, the lack of cell-permeable and low-toxicity small molecules and cognate aptamers that can be exploited as riboswitches and the modest switching performance of mammalian riboswitches have limited their applications. In this study, we systematically optimized the design of a riboswitch that regulates exon skipping via an RNA aptamer that binds ASP2905. We examined two design strategies to modulate the stability of the aptamer base stem that blocks the 5' splice site to fine-tune the riboswitch characteristics. Furthermore, an optimized riboswitch was used to generate a mouse embryonic stem cell line that can be chemically induced to differentiate into myogenic cells by activating Myod1 expression and a human embryonic kidney cell line that can be induced to trigger apoptosis by activating BAX expression. The results demonstrate the tight chemical regulation of transgenes in mammalian cells to control their phenotype without exogenous protein factors.

Knockdown of recA gene expression by artificial small RNAs in Escherichia coli.

Bacterial RecA plays a central role in DNA repair and regulation of the SOS response to DNA damage, and has been suggested as a new antibiotic drug target. To develop a new tool to study RecA function, we engineered artificial small RNAs (sRNAs) that can posttranscriptionally repress RecA expression in Escherichia coli. The artificial sRNAs mimic the bacterial noncoding sRNAs which possess an antisense domain that is partially complementary to the targeted mRNA. We screened a library of artificial sRNAs with a randomized antisense domain and isolated several anti-recA sRNAs that can knockdown the endogenous RecA level in E. coli. The cells expressing the anti-recA sRNAs were found to exhibit phenotypes consistent with RecA repression such as reduced swarming motility and increased susceptibility to ciprofloxacin, a fluoroquinone antibiotic.

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.

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.