• alternative splicing
• aptamer
• arachidonate 5-lipoxygenase
• exon skipping
• synthetic biology
• tetracycline riboswitch

• Humans
• Riboswitch/genetics
• HEK293 Cells
• Arachidonate 5-Lipoxygenase/genetics
• Arachidonate 5-Lipoxygenase/metabolism
• Alternative Splicing/genetics
• Exons/genetics
• Introns/genetics
• Protein Isoforms/genetics
• Protein Isoforms/metabolism
• Gene Expression Regulation
• Tetracycline/pharmacology

• Deutsche Forschungsgemeinschaft

• rna
• riboswitches
• transcriptional regulatory elements

• Riboswitch/genetics
• RNA Folding
• Kinetics
• Aptamers, Nucleotide/metabolism
• Aptamers, Nucleotide/genetics
• Aptamers, Nucleotide/chemistry
• Nucleic Acid Conformation
• RNA, Bacterial/genetics
• RNA, Bacterial/chemistry
• RNA, Bacterial/metabolism
• Ligands
• Gene Expression Regulation, Bacterial

• R01 GM130901 NIGMS NIH HHS
• T32 GM008382 NIGMS NIH HHS
• T32GM008382 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)
• R01GM130901 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)
• U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)
• John Simon Guggenheim Memorial Foundation

• RNA dynamics
• RNA folding
• Riboswitches
• Strand Displacement

• R01 GM130901 NIGMS NIH HHS
• T32 GM008382 NIGMS NIH HHS

• conformational dynamics
• kinetic modelling
• magnesium-driven folding
• riboswitch engineering
• tetracycline-binding aptamer

• Anti-Bacterial Agents/chemistry
• Aptamers, Nucleotide/chemistry
• Ions
• Ligands
• Magnesium/chemistry
• Nucleic Acid Conformation
• Riboswitch
• Tetracycline/chemistry

• biomaterials - cells
• environmental biotechnology
• bioremediation
• synthetic biology
• fluorescent proteins

• Synechococcus/genetics
• Synechococcus/metabolism
• Photosynthesis
• Synthetic Biology/methods
• Bacterial Proteins/genetics
• Bacterial Proteins/metabolism
• Metabolic Engineering/methods
• Hydrogels/metabolism

• P30 NS047101 NINDS NIH HHS
• National Science Foundation (NSF)

• Biomanufacturing
• Cell-free protein synthesis
• In vitro diagnostics
• Metabolic engineering
• Minimal cell
• Synthetic biology

• Bottom-up synthetic biology
• Cell-free protein synthesis
• Minimal cells
• Synthetic cells

• Synthetic Biology/methods
• Biotechnology/methods
• Cell-Free System/metabolism
• Artificial Cells/metabolism

• CRISPR
• RNA chaperones
• RNA metabolism
• RNA regulators
• RNA tool
• ribonucleases
• small non-coding RNAs (ncRNAs)
• synthetic biology
• virulence

• Aptamer
• Enzyme-linked oligonucleotide assay
• Flow cytometry
• Meningitis
• Systematic evolution of ligands by exponential enrichment

• 5′ UTR
• Saccharomyces cerevisiae
• post-transcriptional regulation
• protein synthesis
• regulatory elements

The ability of the cytidine analog Ç_m^f to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Ç_m^f-labeled single- and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Ç_m^f. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Ç_m^f at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.

The quest for a minimal cell not only sheds light on the fundamental principles of life but also brings great advances in related applied fields such as general biotechnology. Minimal cell projects came from the study of a plausible route to the origin of life. Later on, research extended and also referred to the construction of artificial cells, or even more broadly, as in vitro synthetic biology. The cell-free protein synthesis (CFPS) techniques harness the central cellular activity of transcription/translation in an open environment, providing the framework for multiple cellular processes assembling. Therefore, CFPS systems have become the first choice in the construction of the minimal cell. In this review, we focus on the recent advances in the quantitative analysis of CFPS and on its advantage for addressing the bottom-up assembly of a minimal cell and illustrate the importance of systemic chassis behavior, such as stochasticity under a compartmentalized micro-environment.

• cell-free protein synthesis
• chassis
• gene circuits
• micro-compartments
• stochasticity

• Artificial Cells/metabolism
• Cell Compartmentation
• Cell-Free System
• Gene Regulatory Networks
• Protein Biosynthesis
• Stochastic Processes

Multiple mechanisms tightly regulate mRNAs during their transcription, translation, and degradation. Of these, the physical localization of mRNAs to specific cytoplasmic regions is relatively easy to detect; however, linking localization to functional regulatory roles has been more difficult to establish. Historically, Drosophila melanogaster is a highly effective model to identify localized mRNAs and has helped identify roles for this process by regulating various cell activities. The majority of the well-characterized functional roles for localizing mRNAs to sub-regions of the cytoplasm have come from the Drosophila oocyte and early syncytial embryo. At present, relatively few functional roles have been established for mRNA localization within the relatively smaller, differentiated somatic cell lineages characteristic of later development, beginning with the cellular blastoderm, and the multiple cell lineages that make up the gastrulating embryo, larva, and adult. This review is divided into three parts—the first outlines past evidence for cytoplasmic mRNA localization affecting aspects of cellular activity post-blastoderm development in Drosophila. The majority of these known examples come from highly polarized cell lineages such as differentiating neurons. The second part considers the present state of affairs where we now know that many, if not most mRNAs are localized to discrete cytoplasmic regions in one or more somatic cell lineages of cellularized embryos, larvae or adults. Assuming that the phenomenon of cytoplasmic mRNA localization represents an underlying functional activity, and correlation with the encoded proteins suggests that mRNA localization is involved in far more than neuronal differentiation. Thus, it seems highly likely that past-identified examples represent only a small fraction of localization-based mRNA regulation in somatic cells. The last part highlights recent technological advances that now provide an opportunity for probing the role of mRNA localization in Drosophila, moving beyond cataloging the diversity of localized mRNAs to a similar understanding of how localization affects mRNA activity.

• Drosophila melanogaster
• epithelial differentiation
• mRNA localization
• neuronal differentiation
• organelle

• MaxSynBio
• cell-free protein synthesis
• minimal cell
• molecular crowding
• stochasticity
• synthetic biology

• E. coli
• cell sorting
• genetic selection
• riboswitches

Due to their simple architecture and control mechanism, regulatory RNA modules are attractive building blocks in synthetic biology. This is especially true for riboswitches, which are natural ligand-binding regulators of gene expression. The discovery of various tandem riboswitches inspired the design of combined RNA modules with activities not yet found in nature. Riboswitches were placed in tandem or in combination with a ribozyme or temperature-responsive RNA thermometer resulting in new functionalities. Here, we compare natural examples of tandem riboswitches with recently designed artificial RNA regulators suggesting substantial modularity of regulatory RNA elements. Challenges associated with modular RNA design are discussed.

• RNA thermometer
• Riboswitch
• posttranscriptional regulation
• ribozyme
• synthetic biology

Aptamer-protein interacting pairs play a variety of physiological functions and therapeutic potentials in organisms. Rapidly and effectively predicting aptamer-protein interacting pairs is significant to design aptamers binding to certain interested proteins, which will give insight into understanding mechanisms of aptamer-protein interacting pairs and developing aptamer-based therapies.

In this study, an ensemble method is presented to predict aptamer-protein interacting pairs with hybrid features. The features for aptamers are extracted from Pseudo K-tuple Nucleotide Composition (PseKNC) while the features for proteins incorporate Discrete Cosine Transformation (DCT), disorder information, and bi-gram Position Specific Scoring Matrix (PSSM). We investigate predictive capabilities of various feature spaces. The proposed ensemble method obtains the best performance with Youden’s Index of 0.380, using the hybrid feature space of PseKNC, DCT, bi-gram PSSM, and disorder information by 10-fold cross validation. The Relief-Incremental Feature Selection (IFS) method is adopted to obtain the optimal feature set. Based on the optimal feature set, the proposed method achieves a balanced performance with a sensitivity of 0.753 and a specificity of 0.725 on the training dataset, which indicates that this method can solve the imbalanced data problem effectively. To evaluate the prediction performance objectively, an independent testing dataset is used to evaluate the proposed method. Encouragingly, our proposed method performs better than previous study with a sensitivity of 0.738 and a Youden’s Index of 0.451.

These results suggest that the proposed method can be a potential candidate for aptamer-protein interacting pair prediction, which may contribute to finding novel aptamer-protein interacting pairs and understanding the relationship between aptamers and proteins.

The online version of this article (doi:10.1186/s12859-016-1087-5) contains supplementary material, which is available to authorized users.

• Aptamer-protein interacting pairs
• Ensemble method
• Hybrid features
• Imbalanced data problem

Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers–called P12-31—is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies.

In this paper, we describe a point-of-care biosensor design. The uniqueness of our design is in its capability for detecting a wide variety of target biomolecules and the simplicity of nanoparticle enhanced electrical detection. The electrical properties of interdigitated electrodes (IDEs) and the mechanism for gold nanoparticle-enhanced impedance-based biosensor systems based on these electrodes are simulated using COMSOL Multiphysics software. Understanding these properties and how they can be affected is vital in designing effective biosensor devices. Simulations were used to show electrical screening develop over time for IDEs in a salt solution, as well as the electric field between individual digits of electrodes. Using these simulations, it was observed that gold nanoparticles bound closely to IDEs can lower the electric field magnitude between the digits of the electrode. The simulations are also shown to be a useful design tool in optimizing sensor function. Various different conditions, such as electrode dimensions and background ion concentrations, are shown to have a significant impact on the simulations.

• COMSOL
• biosensor
• gold nanoparticles
• interdigitated electrodes

• Biosensing Techniques/instrumentation
• Computer Simulation
• Electric Impedance
• Electrodes
• Equipment Design
• Gold/chemistry
• Metal Nanoparticles/chemistry

We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein–RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5′ untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells.

• NMR spectroscopy
• RNA structures
• aptamers
• gene expression
• riboswitches
• synthetic biology

• RNA
• chemical biology
• photocontrol
• riboswitches
• synthetic biology

• NMR
• PreQ(0)
• Queuine
• Queuosine
• X-ray crystallography
• tRNA modification

• RNA
• gene expression
• gene technology
• inducible gene expression
• optogenetics

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

We engineered small molecule responsive allosteric ribozymes based on the genomic hepatitis delta virus (HDV) ribozyme by replacing the P4-L4 stem-loop with an RNA aptamer through a connector stem. When embedded in the 3′ untranslated region of a reporter gene mRNA, these RNA devices enabled regulation of cis-gene expression by theophylline and guanine by up to 29.5-fold in mammalian cell culture. Furthermore, a NOR logic gate device was constructed by placing two engineered ribozymes in tandem, demonstrating the modularity of the RNA devices. The significant improvement in the regulatory dynamic range (ON/OFF ratio) of the RNA devices based on the HDV ribozyme should provide new opportunities for practical applications.

• synthetic biology
• tuning
• tunable
• response curves
• biological rates

• Models, Biological
• Synthetic Biology

• RNA aptamer
• RNA protein interaction
• Synthetic biology
• Tet repressor
• Transcription regulation

• Aptamers, Nucleotide/chemistry
• Aptamers, Nucleotide/genetics
• Base Sequence
• Escherichia coli/genetics
• Gene Expression
• Molecular Sequence Data
• Plasmids/genetics
• Protein Binding
• RNA/analysis
• RNA/biosynthesis
• RNA/genetics
• RNA, Untranslated/analysis
• RNA, Untranslated/biosynthesis
• RNA, Untranslated/genetics
• Tetracycline/metabolism
• Tetracycline/pharmacology
• Transcription Factors/chemistry
• Transcription, Genetic

Aptamers are single-stranded oligonucleotides that bind to targets with high affinity and selectivity. Their use as molecular recognition elements has emerged as a viable approach for biosensing, diagnostics, and therapeutics. Despite this potential, relatively few aptamers exist that bind to small molecules. Small molecules are important targets for investigation due to their diverse biological functions as well as their clinical and commercial uses. Novel, effective molecular recognition probes for these compounds are therefore of great interest. This paper will highlight the technical challenges of aptamer development for small molecule targets, as well as the opportunities that exist for their application in biosensing and chemical biology.

Small GTP-binding proteins of the ADP-ribosylation factor (Arf) family control various cell functional responses including protein transport and recycling between different cellular compartments, phagocytosis, proliferation, cytoskeletal remodelling, and migration. The activity of Arfs is tightly regulated. GTPase-activating proteins (GAPs) inactivate Arfs by stimulating GTP hydrolysis, and guanine nucleotide exchange factors (GEFs) stimulate the conversion of inactive GDP-bound Arf to the active GTP-bound conformation. There is increasing evidence that Arf small GTPases contribute to cancer growth and invasion. Increased expression of Arf6 and of Arf-GEPs, or deregulation Arf-GAP functions have been correlated with enhanced invasive capacity of tumor cells and metastasis. The spatiotemporal specificity of Arf activation is dictated by their GEFs that integrate various signals in stimulated cells. Brefeldin A (BFA), which inactivates a subset of Arf-GEFs, has been very useful for assessing the function of Golgi-localized Arfs. However, specific inhibitors to investigate the individual function of BFA-sensitive and insensitive Arf-GEFs are lacking. In recent years, specific screens have been developed, and new inhibitors with improved selectivity and potency to study cell functional responses regulated by BFA-sensitive and BFA-insensitive Arf pathways have been identified. These inhibitors have been instrumental for our understanding of the spatiotemporal activation of Arf proteins in cells and demonstrate the feasibility of developing small molecules interfering with Arf activation to prevent tumor invasion and metastasis.

• Golgi
• Vesicular transport
• Migration
• Adhesion
• Actin

• Allosteric Regulation
• Aptamers, Nucleotide/genetics
• Aptamers, Nucleotide/metabolism
• Base Sequence
• Genes, Essential/genetics
• Genes, Reporter/genetics
• HeLa Cells
• Humans
• Protein Biosynthesis
• RNA Splicing/genetics
• RNA Stability
• RNA, Catalytic/chemistry
• RNA, Catalytic/genetics
• RNA, Catalytic/metabolism
• Riboswitch/genetics
• Saccharomyces cerevisiae/genetics
• Synthetic Biology/methods

Obtaining homogeneous population of natively folded RNAs is a crippling problem encountered when preparing RNAs for structural or enzymatic studies. Most of the traditional methods that are employed to prepare large quantities of RNAs involve procedures that partially denature the RNA. Here, we present a simple strategy using ‘click’ chemistry to couple biotin to a ‘caged’ photocleavable (PC) guanosine monophosphate (GMP) in high yield. This biotin-PC GMP, accepted by T7 RNA polymerase, has been used to transcribe RNAs ranging in size from 27 to 527 nt. Furthermore we show, using an in-gel fluorescence assay, that natively prepared 160 and 175 kDa minimal group II intron ribozymes have enhanced catalytic activity over the same RNAs, purified via denaturing conditions and refolded. We conclude that large complex RNAs prepared by non-denaturing means form a homogeneous population and are catalytically more active than those prepared by denaturing methods and subsequent refolding; this facile approach for native RNA preparation should benefit synthesis of RNAs for biophysical and therapeutic applications.