Engineered riboswitches as novel tools in molecular biology

During the last years the great importance of RNA for regulating gene expression in all organisms has become obvious. Consequently, several recent approaches aim to utilize the outstanding chemical properties of RNA to develop artificial RNA regulators for conditional gene expression systems. A combination of rational design, in vitro selection and in vivo screening systems has been used to create a versatile set of RNA based molecular switches. These tools rely on diverse mechanisms and exhibit activity in several organisms. In this review, we summarize recent developments in the application of engineered riboswitches for gene regulation in vivo.     

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.     

RNA synthetic biology

RNA molecules play important and diverse regulatory roles in the cell by virtue of their interaction with other nucleic acids, proteins and small molecules. Inspired by this natural versatility, researchers have engineered RNA molecules with new biological functions. In the last two years efforts in synthetic biology have produced novel, synthetic RNA components capable of regulating gene expression in vivo largely in bacteria and yeast, setting the stage for scalable and programmable cellular behavior. Immediate challenges for this emerging field include determining how computational and directed-evolution techniques can be implemented to increase the complexity of engineered RNA systems, as well as determining how such systems can be broadly extended to mammalian systems. Further challenges include designing RNA molecules to be sensors of intracellular and environmental stimuli, probes to explore the behavior of biological networks and components of engineered cellular control systems.     

Synthetic RNA circuits

Natural and engineered RNA 'parts' can perform a variety of functions, including hybridizing to targets, binding ligands and undergoing programmed conformational changes, and catalyzing reactions. These RNA parts can in turn be assembled into synthetic genetic circuits that regulate gene expression by acting either in cis or in trans on mRNAs. As more parts are discovered and engineered, it should be increasingly possible to create synthetic RNA circuits that are able to carry out complex logical operations in cells, either superimposed on or autonomous to extant gene regulation.     

Opportunities in the design and application of RNA for gene expression control

The past decade of synthetic biology research has witnessed numerous advances in the development of tools and frameworks for the design and characterization of biological systems. Researchers have focused on the use of RNA for gene expression control due to its versatility in sensing molecular ligands and the relative ease by which RNA can be modeled and designed compared to proteins. We review the recent progress in the field with respect to RNA-based genetic devices that are controlled through small molecule and protein interactions. We discuss new approaches for generating and characterizing these devices and their underlying components. We also highlight immediate challenges, future directions and recent applications of synthetic RNA devices in engineered biological systems.     

Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression

Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA–RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.     

Engineered riboswitches: Expanding researchers' toolbox with synthetic RNA regulators

Riboswitches are natural RNA-based genetic switches that sense small-molecule metabolites and regulate in response the expression of the corresponding metabolic genes. Within the last years, several engineered riboswitches have been developed that act on various stages of gene expression. These switches can be engineered to respond to any ligand of choice and are therefore of great interest for synthetic biology. In this review, we present an overview of engineered riboswitches and discuss their application in conditional gene expression systems. We will provide structural and mechanistic insights and point out problems and recent trends in the development of engineered riboswitches.     

Engineering dynamic cell cycle control with synthetic small molecule-responsive RNA devices

Background

The cell cycle plays a key role in human health and disease, including development and cancer. The ability to easily and reversibly control the mammalian cell cycle could mean improved cellular reprogramming, better tools for studying cancer, more efficient gene therapy, and improved heterologous protein production for medical or industrial applications.

Results

We engineered RNA-based control devices to provide specific and modular control of gene expression in response to exogenous inputs in living cells. Specifically, we identified key regulatory nodes that arrest U2-OS cells in the G0/1 or G2/M phases of the cycle. We then optimized the most promising key regulators and showed that, when these optimized regulators are placed under the control of a ribozyme switch, we can inducibly and reversibly arrest up to ~80 % of a cellular population in a chosen phase of the cell cycle. Characterization of the reliability of the final cell cycle controllers revealed that the G0/1 control device functions reproducibly over multiple experiments over several weeks.

Conclusions

To our knowledge, this is the first time synthetic RNA devices have been used to control the mammalian cell cycle. This RNA platform represents a general class of synthetic biology tools for modular, dynamic, and multi-output control over mammalian cells.

     

Molecular methods for environmental monitoring and containment of genetically engineered microorganisms

Plans to introduce genetically engineered microorganisms into the environment has led to concerns over safety and has raised questions about how to detect and to contain such microorganisms. Specific gene sequences, such as lacZ, have been inserted into genetically engineered microorganisms to permit their phenotypic detection. Molecular methods have been developed based upon recovery of DNA from environmental samples and gene probe hybridization to specific diagnostic gene sequences for the specific detection of genetically engineered microorganisms. DNA amplification using the polymerase chain reaction has been applied to enhance detection sensitivity so that single gene targets can be detected. Detection of messenger RNA has permitted the monitoring of gene expression in the environment. The use of reporter genes, such as the lux gene for bioluminescence, likewise has permitted the observation of gene expression. Conditional lethal constructs have been developed as models for containment of genetically engineered microorganisms. Suicide vectors, based upon the hok gene have been developed as model containment systems.     

MicroRNA 与垂体腺瘤关系的研究进展

MicroRNA(miRNA)是新发现的一类特殊的小分子RNA,在基因的转录后调控中扮演重要的角色.研究发现很多良恶性肿瘤在发生发展过程中都伴有miRNA的异常表达,所以miRNA的转录后调控机制以及在肿瘤成瘤过程中的作用逐渐成为当下研究的热点,本文就目前垂体腺瘤与miRNA的研究现状做以简要概述.