Scalable control of stem cell fate by riboswitch-regulated RNA viral vector without genomic integration.

Transgene expression in stem cells is a powerful means of regulating cellular properties and differentiation into various cell types. However, existing vectors for transgene expression in stem cells suffer from limitations such as the need for genomic integration, the transient nature of gene expression, and the inability to temporally regulate transgene expression, which hinder biomedical and clinical applications. Here we report a new class of RNA virus-based vectors for scalable and integration-free transgene expression in mouse embryonic stem cells (mESCs).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 RNA-ligand interaction and respond by regulating protein synthesis in cell-free protein synthesis systems.

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.

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.

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.

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.

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.

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.

Gas-Phase Synthesis for Label-Free Biosensors: Zinc-Oxide Nanowires Functionalized with Gold Nanoparticles.

Metal oxide semiconductor nanowires have important applications in label-free biosensing due to their ease of fabrication and ultralow detection limits. Typically, chemical functionalization of the oxide surface is necessary for specific biological analyte detection. We instead demonstrate the use of gas-phase synthesis of gold nanoparticles (Au NPs) to decorate zinc oxide nanowire (ZnO NW) devices for biosensing applications.

Development of a histamine aptasensor for food safety monitoring.

Histamine produced by bacteria through decarboxylation of histidine in spoiled foods such as fish is known to cause food poisoning. Therefore, accurate and facile measurement of histamine is of practical importance. Using the recently discovered RNA aptamer that specifically recognizes histamine (A1-949 aptamer), we developed an aptasensor based on the structure-switching mechanism.