A FACS‐Based Approach to Engineering Artificial Riboswitches

Fowler, Casey C.; Brown, Eric D.; Li, Yingfu

doi:10.1002/cbic.200700713

Cited by 66 publications

(51 citation statements)

References 45 publications

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“…Furthermore, the reporter gene in use seems to have an impact on the result as well. Fowler et al 40 and K€ otter et al 41 observed that the insertion of secondary structure elements in the 5 0 -UTR of a GFP gene can lead to a reduction in gene expression and, consequently, fluorescence intensity. Obviously, some of the riboswitch elements provoke a similar reduction.…”

Section: Increasing the Activation Ratio By Tandem And Tridem Constructsmentioning

confidence: 99%

Design criteria for synthetic riboswitches acting on transcription

et al. 2015

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Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

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Section: Increasing the Activation Ratio By Tandem And Tridem Constructsmentioning

confidence: 99%

Design criteria for synthetic riboswitches acting on transcription

et al. 2015

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“…Notably, insertion of aptamers into the 5′-UTRs of bacterial or eukaryotic mRNAs (9)(10)(11)(12), splice sites within introns of eukaryotic pre-mRNA (13,14), or coupling of aptamers to ribozymes (15,16) have shown significant potential for small-molecule modulation of gene expression. However, there are currently very few aptamers, generated by in vitro selection, that are selective for ligands with desirable physicochemical and pharmacokinetic properties (8).…”

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confidence: 99%

Reengineering orthogonally selective riboswitches

Dixon

Duncan²,

Geerlings³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

151

163

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The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule-dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic "nonnatural" small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.

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“…Another automated approach that was demonstrated to be highly amenable with structure-switching sensors is fluorescence-activated cell sorting (FACS), a method that can sort fluorescent cells faster than 10 4 cells a second [44]. Juewen Liu and colleagues immobilized the anti-ATP structure-switching aptamer onto magnetic microparticles, and implemented FACS to sort out microparticles that underwent target-induced structure-switching (high fluorescence) from those that retained the duplex of fluorescein-labeled aptamer and QDNA (low fluorescence) [45].…”

Section: Structure-switching Dna Aptamers In Fluorescent Sensorsmentioning

confidence: 99%

Exploration of Structure-Switching in the Design of Aptamer Biosensors

Lau

2013

Advances in Biochemical Engineering/Biotechnology

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The process of "structure-switching" enables biomolecular switches to function as effective biosensing tools. Biomolecular switches can be activated or inactivated by binding to a specific target that triggers a precise conformational change in the biomolecules involved. Although many examples of aptamer-based biomolecular switches can be found in nature, substantial effort has been made in the last decade to engineer structure-switching aptamer sensors by coupling aptamers to a signal transduction method to generate a readout signal upon target binding to the aptamer domain. This chapter focuses on the progress of research on engineered structure-switching aptamer sensors. We begin by discussing the origin of the structure-switching aptamer design, highlight the key developments of structure-switching DNA aptamers for fluorescence-, electrochemistry-, and colorimetry-based detection, and introduce our recent efforts in exploring RNA aptamers to create structure-switching molecular sensors.

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A FACS‐Based Approach to Engineering Artificial Riboswitches

Cited by 66 publications

References 45 publications

Design criteria for synthetic riboswitches acting on transcription

Design criteria for synthetic riboswitches acting on transcription

Reengineering orthogonally selective riboswitches

Exploration of Structure-Switching in the Design of Aptamer Biosensors

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