Design Principles for Riboswitch Function

Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence–function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands.     

Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments

Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms.     

Riboswitches in unexpected places--a synthetic riboswitch in a protein coding region

In natural and engineered systems, cis-RNA regulatory elements such as riboswitches are typically found within untranslated regions rather than within the protein coding sequences of genes. However, RNA sequences with important regulatory roles can exist within translated regions. Here, we present a synthetic riboswitch that is encoded within the translated region of a gene and represses Escherichia coli gene expression greater than 25-fold in the presence of a small-molecule ligand. The ability to encode riboswitches within translated regions as well as untranslated regions provides additional opportunities for creating new genetic control elements. Furthermore, evidence that a riboswitch can function in the translated region of a gene suggests that future efforts to identify natural riboswitches should consider this possibility.     

Riboswitch regulation mechanisms: RNA,metabolites and regulatory proteins

Riboswitches are RNA sensors that have been shown to modulate the expression of downstream genes by altering their structure upon metabolite binding. Riboswitches are unique among cellular regulators in that metabolite detection is strictly performed using RNA interactions with the sensed metabolite and in which no regulatory protein is needed to mediate the interaction. However, recent studies have shed light on riboswitch control mechanisms relying on protein regulators to harness metabolite binding for the mediation of gene expression, thereby increasing the range of cellular factors involved in riboswitch regulation. The interaction between riboswitches and proteins adds another level of evolutionary pressure as riboswitches must maintain key residues for metabolite detection, structural switching and protein binding sites. Here, we review regulatory mechanisms involving Escherichia coli riboswitches that have recently been shown to rely on regulatory proteins. We also discuss the implication of such protein-based riboswitch regulatory mechanisms for genetic regulation.     

Cell-free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab-on-chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell-free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell-cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell-free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.     

新型基因表达调控元件——人工核糖开关的构建及筛选

核糖开关作为一种新发现的RNA元件,可以高效、准确、快速地执行基因调控任务,且免疫原性低,有可能在将来以顺式模块的方式应用于未来的基因治疗。近年来已经成功构建了多种人造核糖开关,构建方法主要是利用人工适体元件与基因表达调控元件组装,或者是在天然核糖开关基础上进行改造。文中全面综述了涉及人工核糖开关设计及筛选的技术,讨论了可以用于哺乳细胞、响应非天然配体信号、调控特征为热力学和动力学控制的核糖开关的设计新策略,并对核糖开关的筛选构建策略及其在基因治疗及新型药物开发领域的应用前景进行了展望。尽管目前将核糖开关设计成为功能强大的新型基因调控系统还面临很大的困难,但通过构效关系的研究、计算机辅助设计、体外筛选及细胞内筛选技术、高通量优化筛选等技术的综合应用,核糖开关一定可以成为有力的基因调控工具,如能成功应用则可大大促进基因治疗临床化的进程。     

Development of cell-free protein synthesis platforms for disulfide bonded proteins

The use of cell-free protein synthesis (CFPS) for recombinant protein production is emerging as an important technology. For example, the openness of the cell-free system allows control of the reaction environment to promote folding of disulfide bonded proteins in a rapid and economically feasible format. These advantages make cell-free protein expression systems particularly well suited for producing patient specific therapeutic vaccines or antidotes in response to threats from natural and man-made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. In this work we assess the versatility of modern cell-free methods, optimize expression and folding parameters, and highlight the importance of rationally designed plasmid templates for producing mammalian secreted proteins, fusion proteins, and antibody fragments in our E. coli-based CFPS system. Two unique CFPS platforms were established by developing standardized extract preparation protocols and generic cell-free reaction conditions. Generic reaction conditions enabled all proteins to express well with the best therapeutic protein yield at 710 microg/mL, an antibody fragment at 230 microg/mL, and a vaccine fusion protein at 300 microg/mL; with the majority correctly folded. Better yields were obtained when cell-free reaction conditions were optimized for each protein. Establishing general CFPS platforms enhances the potential for cell-free protein synthesis to reliably produce complex protein products at low production and capital costs with very rapid process development timelines.     

小分子靶标的核糖开关生物传感器研究进展

小分子化合物种类繁多，在众多生化过程中发挥关键作用，具有重要的检测意义与价值,其快速灵敏可视化检测技术的开发是当前的研究热点。基于核糖开关的生物传感器因具有识别特性高、操作简便、成本低等优势，为小分子检测提供了一条新途径。对核糖开关的来源、构成、调控机制、体内外筛选，特别是对基于小分子靶标的核糖开关生物传感器分类进行了介绍，并从核糖开关的筛选、裁剪、理性设计、核糖开关无细胞传感器的应用等几个方向提出了展望，以期为小分子靶标的核糖开关生物传感器的发展和应用提供理论依据。     

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.     

Hydrofoam and oxygen headspace bioreactors improve cell-free therapeutic protein production yields through enhanced oxygen transport

Protein therapeutics are powerful tools in the fight against diabetes, cancers, growth disorders, and many other debilitating diseases. However, availability is limited due to cost and complications of production from living organisms. To make life-saving protein therapeutics more available to the world, the possibility of magistral or point-of-care protein therapeutic production has gained focus. The recent invention and optimization of lyophilized “cell-free” protein synthesis reagents and its demonstrated ability to produce highly active versions of FDA-approved cancer therapeutics have increased its potential for low-cost, single-batch, magistral medicine. Here we present for the first time the concept of increased oxygen mass transfer in small-batch, cell-free protein synthesis (CFPS) reactions through air-water foams. These “hydrofoam” reactions increased CFPS yields by up to 100%. Contrary to traditional protein synthesis using living organisms, where foam bubbles cause cell-lysis and production losses, hydrofoam CFPS reactions are “cell-free” and better tolerate foaming. Simulation and experimental results suggest that oxygen transfer is limiting in even small volume batch CFPS reactors and that the hydrofoam format improved oxygen transfer. This is further supported by CFPS reactions achieving higher yields when oxygen gas replaces air in the headspace of batch reactions. Improving CFPS yields with hydrofoam reduces the overall cost of biotherapeutic production, increasing availability to the developing world. Beyond protein therapeutic production, hydrofoam CFPS could also be used to enhance other CFPS applications including biosensing, biomanufacturing, and biocatalysis.     

Purine sensing by riboswitches.

Structured mRNA elements called riboswitches control gene expression by binding to small metabolites. Over a dozen riboswitch classes have been characterized that target a broad range of molecules and vary widely in size and secondary structure. Four of the known riboswitch classes recognize purines or modified purines. Three of these classes are closely related in conserved sequence and secondary structure, but members of these classes selectively recognize guanine, adenine or 2'-deoxyguanosine. Members of the fourth riboswitch class adopt a distinct structure to form a selective binding pocket for the guanine analogue preQ(1) (7-aminomethyl-7-deazaguanine). All four classes of purine-sensing riboswitches are most likely to recognize their respective metabolites by utilizing a riboswitch residue to make a canonical Watson-Crick base-pair with the ligand. This review will provide a summary of the purine-sensing riboswitches, as well as discuss the complex functions and applications of these RNAs.     

Purine sensing by riboswitches

Structured mRNA elements called riboswitches control gene expression by binding to small metabolites. Over a dozen riboswitch classes have been characterized that target a broad range of molecules and vary widely in size and secondary structure. Four of the known riboswitch classes recognize purines or modified purines. Three of these classes are closely related in conserved sequence and secondary structure, but members of these classes selectively recognize guanine, adenine or 2'-deoxyguanosine. Members of the fourth riboswitch class adopt a distinct structure to form a selective binding pocket for the guanine analogue preQ(1) (7-aminomethyl-7-deazaguanine). All four classes of purine-sensing riboswitches are most likely to recognize their respective metabolites by utilizing a riboswitch residue to make a canonical Watson-Crick base-pair with the ligand. This review will provide a summary of the purine-sensing riboswitches, as well as discuss the complex functions and applications of these RNAs.     

Riboswitches: small-molecule recognition by gene regulatory RNAs

Riboswitches demonstrate the ability of highly structured RNA molecules to recognize small-molecule metabolites with high specificity and subsequently harness the binding energy for the control of gene expression. Crystal structures have now been determined for the metabolite-binding domains of riboswitches that respond to purines, thiamine pyrophosphate and S-adenosylmethionine, as well as for the glmS ribozyme, a catalytic riboswitch that is activated by the metabolite glucosamine-6-phosphate. In addition to these riboswitch structures, a solution NMR structure has been reported for a ribosensor that regulates heat shock genes in response to changes in temperature. These studies reveal the structural basis of the remarkable selectivity of riboswitches and, in conjunction with biochemical and biophysical measurements, provide a framework for detailed mechanistic understanding of riboswitch-mediated modulation of gene expression.     

Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism

Riboswitches regulate gene expression via ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. By unfolding and refolding single add adenine riboswitch molecules in an optical trap, an integrated picture of the folding was developed and related to the regulatory mechanism. Force-extension curves (FECs) and constant-force folding trajectories measured on the aptamer alone revealed multiple partially-folded states, including several misfolded states not on the native folding pathway. All states were correlated to key structural components and interactions within hierarchical folding pathways. FECs of the full-length riboswitch revealed that the thermodynamically stable conformation switches upon ligand binding from a structure repressing translation to one permitting it. Along with rapid equilibration of the two structures in the absence of adenine, these results support a thermodynamically-controlled regulatory mechanism, in contrast with the kinetic control of the closely-related pbuE adenine riboswitch. Comparison of the folding of these riboswitches revealed many similarities arising from shared structural features but also essential differences related to their different regulatory mechanisms.     

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology.     

Ligand binding and gene control characteristics of tandem riboswitches in Bacillus anthracis

Most riboswitches are composed of a single metabolite-binding aptamer and a single expression platform that function together to regulate genes in response to changing metabolite concentrations. In rare instances, two aptamers or sometimes two complete riboswitches reside adjacent to each other in untranslated regions (UTRs) of mRNAs. We have examined an example of a tandem riboswitch in the Gram-positive bacterium Bacillus anthracis that includes two complete riboswitches for thiamine pyrophosphate (TPP). Unlike other complex riboswitch systems described recently, tandem TPP riboswitches do not exhibit cooperative ligand binding and do not detect two different types of metabolites. In contrast, both riboswitches respond independently to TPP and are predicted to function in concert to mimic the more "digital" gene control outcome observed when two aptamers bind ligands cooperatively. Our findings further demonstrate that simple gene control elements made only of RNA can be assembled in different architectures to yield more complex gene control outcomes.