Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Dual-Selection for Evolution of In Vivo Functional Aptazymes as Riboswitch Parts
We describe a broadly applicable method for coupling a novel, newly selected aptamer to a ribozyme to generate functional aptazymes via in vitro and in vivo selection. To illustrate this approach, we describe experimental procedures for selecting aptazymes assembled from aptamers that bind p-amino-phenylalanine and a hammerhead ribozyme. Because this method uses selection, it does not rely on sequence-specific design and thus should be generalizable for the generation of in vivo operational aptazymes that respond to any targeted molecules. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
In Vivo Screening for Aptazyme-Based Bacterial Riboswitches
In many synthetic biology applications, modular and easily accessible tools for controlling gene expression are required. In addition, in vivo biosensors and diagnostic devices will become more important in the future to allow for noninvasive determination of protein, ion, or small molecule metabolite levels. In recent years synthetic RNA-based switches have been developed to act as signal transducers to convert a binding event of a small molecule (input) into a detectable output. Their modular design allows the development of a variety of molecular switches to be used in biochemical assays or inside living cells. RNA swit...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Construction of Ligand-Responsive MicroRNAs that Operate Through Inhibition of Drosha Processing
MicroRNAs (miRNAs) offer powerful tools for targeted gene silencing in almost all eukaryotes. These tools have received considerable attention for their utility in both fundamental genetic studies and as therapeutic agents. Rendering individual microRNAs responsive to endogenous or exogenously applied molecules (or ligands) can improve the stringency of silencing and can mediate autonomous control. This chapter describes the construction of ligand-responsive miRNAs that undergo reduced processing and subsequent gene silencing when bound by the recognized ligand. Following a simple set of rules, the engineered microRNAs can...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Engineered Riboswitch as a Gene-Regulatory Platform for Reducing Antibiotic Resistance
Antibiotic resistance (AR), the ability of a microorganism to withstand the effects of antibiotics, is a growing and increasingly serious global public health problem. Enzymatic activation of antibiotics though the production of β-lactamase is one of the main mechanisms causing AR. Synthetic riboswitch containing aptazyme is constructed in E. coli to regulate the expression of β-lactamase through small molecule–aptamer interactions, which sharply reduces the antibiotic resistance of the engineered bacteria. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Computational Design of RNA Libraries for In Vitro Selection of Aptamers
We present here a computational approach for designing a starting library of RNA sequences with increased formation of complex structural motifs and enhanced affinity to a desired target molecule. Our approach consists of two steps: (1) generation of RNA sequences based on customized patterning of nucleotides with increased probability of forming a base pair and (2) a high-throughput virtual screening of the generated library to select aptamers with binding affinity to a small-molecule target. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
A Three-Dimensional Design Strategy for a Protein-Responsive shRNA Switch
We have recently developed synthetic short hairpin RNA (shRNA) switches that respond to intracellular proteins and control the expression of target genes in mammalian cells (Kashida et al. Nucleic Acids Res 40:9369–9378, 2012; Saito et al. Nat Commun 2:160, 2011). Here, we describe a method for the three-dimensional (3D) design of a protein-responsive shRNA switch that employs modeling software and known 3D structures of RNA–protein complexes. We were able to predict the effect of steric hindrance between the Dicer enzyme and shRNA-binding protein in silico by superimposing the 3D model of the shRNA switch on D...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Development of Photoswitchable RNA Aptamer–Ligand Complexes
Photoresponsive artificial riboswitch has the potential to offer a de novo method for spatiotemporal control of gene expression in living cells. Because, even today, it is difficult to design a small molecule binding to a specific RNA sequence, generating an artificial riboswitch that possesses highly specific affinity to a ligand of interest basically depends on in vitro selection procedure where a variety of RNA–ligand complexes can be obtained in established methods. Here, we describe the protocol for in vitro aptamer selection against a photoresponsive peptide ligand containing azobenzene moiety that undergoes ph...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
In Vitro Selection of RNA Aptamers for a Small-Molecule Dye
Artificial riboswitches can be generated by functional coupling of an RNA aptamer for synthetic small molecule to an expression platform. RNA aptamers that can bind strongly and selectively to their target are feasible to be used for obtaining more potent artificial riboswitches. In this chapter, we describe tips and notes for in vitro selection of RNA aptamers targeting synthetic small molecules. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Identification of RNA Aptamers Against Recombinant Proteins with a Hexa-Histidine Tag
Artificial riboswitches that respond to the concentrations of intracellular proteins are a promising tool with a variety of applications. They can be designed and engineered using existing RNA aptamers that target proteins. Aptamers are generated via an iterative selection–amplification process, known as systematic evolution of ligands by exponential enrichment (SELEX). This chapter describes a SELEX procedure for the identification of RNA aptamers against hexa-histidine-tagged proteins. For the efficient enrichment of higher affinity aptamers, the selection stringency should be gradually increased. Undesired RNA spe...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
FRET-Based Optical Assay for Selection of Artificial Riboswitches
Artificial riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules and, therefore, can be useful tools to reprogram cellular behavior for different applications. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer with a randomized expression platform followed by in vivo selection and screening. Here, we describe an in vivo selection and screening technique to discover artificial riboswitches in E. coli cells that is based on TEV protease–FRET substrate reporter system. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Artificial Riboswitch Selection: A FACS-Based Approach
Riboswitches have a number of characteristics that make them ideal regulatory elements for a wide range of synthetic biology applications. To maximize their utility, methods are required to create custom riboswitches de novo or to modify existing riboswitches to suit specific experimental needs. This chapter describes such a method, which exploits fluorescence-activated cell sorting (FACS) to quickly and efficiently sort through large libraries of riboswitch-like sequences to identify those with the desired activity. Suggestions for the experimental setup are provided, along with detailed protocols for testing and optimizi...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Portable Two-Way Riboswitches: Design and Engineering
Riboswitches are RNA-based regulatory devices that mediate ligand-dependent control of gene expression. However, there has been limited success in rationally designing riboswitches. Moreover, most previous riboswitches are confined to a particular gene and only perform one-way regulation. Here, we describe a library screening strategy for efficient creation of ON riboswitches of lacI of Escherichia coli. An ON riboswitch of lacI is then integrated with the lac promoter, generating a hybrid device to achieve portable sequential OFF-and-ON gene regulation. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Dual Genetic Selection of Synthetic Riboswitches in Escherichia coli
This chapter describes a genetic selection strategy to engineer synthetic riboswitches that can chemically regulate gene expression in Escherichia coli. Riboswitch libraries are constructed by randomizing the nucleotides that potentially comprise an expression platform and fused to the hybrid selection/screening marker tetA–gfpuv. Iterative ON and OFF selections are performed under appropriate conditions that favor the survival or the growth of the cells harboring the desired riboswitches. After the selection, rapid screening of individual riboswitch clones is performed by measuring GFPuv fluorescence without subclon...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Generation of Orthogonally Selective Bacterial Riboswitches by Targeted Mutagenesis and In Vivo Screening
Riboswitches are naturally occurring RNA-based genetic switches that control gene expression in response to the binding of small-molecule ligands, typically through modulation of transcription or translation. Their simple mechanism of action and the expanding diversity of riboswitch classes make them attractive targets for the development of novel gene expression tools. The essential first step in realizing this potential is to generate artificial riboswitches that respond to nonnatural, synthetic ligands, thereby avoiding disruption of normal cellular function. Here we describe a strategy for engineering orthogonally sele...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Sample Preparation Methods for LC-MS-Based Global Aqueous Metabolite Profiling
Metabolite extraction is a key step in metabolomic analyses, particularly for untargeted studies. The extraction determines the types of metabolites that will be detected and the analytical platform to be used. In this chapter we describe two protocols aimed at detecting polar metabolites from biological samples; the first is aimed at detecting reduced species by LC/MS, and the second satisfies the requirements for both NMR and LC/MS analysis simultaneously. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - January 1, 2014 Category: Biochemistry Source Type: news
