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Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells.

ABSTRACT: Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient cis-acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an aptazymes's activity by integrating three characteristics of communication-module bases: hydrogen bonding, base stacking, and distance to the enzymatic core. We validated the power and generality of this approach by designing aptazymes responsive to three distinct ligands, each with markedly wider dynamic ranges than any previously reported. These aptayzmes efficiently regulated adeno-associated virus (AAV)-vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches. Our approach enables efficient, protein-independent control of gene expression by a range of small molecules.

SUBMITTER: Zhong G

PROVIDER: S-EPMC5130294 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells.

Zhong Guocai G Wang Haimin H Bailey Charles C CC Gao Guangping G Farzan Michael M

eLife 20161102

Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient <i>cis</i>-acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an ...[more]

PMID: 27805569

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Project description:Background¹³C-Metabolic flux analysis (¹³C-MFA) is a standard technique to probe cellular metabolism and elucidate in vivo metabolic fluxes. 13C-Tracer selection is an important step in conducting ¹³C-MFA, however, current methods are restricted to trial-and-error approaches, which commonly focus on an arbitrary subset of the tracer design space. To systematically probe the complete tracer design space, especially for complex systems such as mammalian cells, there is a pressing need for new rational approaches to identify optimal tracers.ResultsRecently, we introduced a new framework for optimal ¹³C-tracer design based on elementary metabolite units (EMU) decomposition, in which a measured metabolite is decomposed into a linear combination of so-called EMU basis vectors. In this contribution, we applied the EMU method to a realistic network model of mammalian metabolism with lactate as the measured metabolite. The method was used to select optimal tracers for two free fluxes in the system, the oxidative pentose phosphate pathway (oxPPP) flux and anaplerosis by pyruvate carboxylase (PC). Our approach was based on sensitivity analysis of EMU basis vector coefficients with respect to free fluxes. Through efficient grouping of coefficient sensitivities, simple tracer selection rules were derived for high-resolution quantification of the fluxes in the mammalian network model. The approach resulted in a significant reduction of the number of possible tracers and the feasible tracers were evaluated using numerical simulations. Two optimal, novel tracers were identified that have not been previously considered for ¹³C-MFA of mammalian cells, specifically [2,3,4,5,6-¹³C]glucose for elucidating oxPPP flux and [3,4-¹³C]glucose for elucidating PC flux. We demonstrate that ¹³C-glutamine tracers perform poorly in this system in comparison to the optimal glucose tracers.ConclusionsIn this work, we have demonstrated that optimal tracer design does not need to be a pure simulation-based trial-and-error process; rather, rational insights into tracer design can be gained through the application of the EMU basis vector methodology. Using this approach, rational labeling rules can be established a priori to guide the selection of optimal ¹³C-tracers for high-resolution flux elucidation in complex metabolic network models.

Dataset Information

Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells.

Publications

Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells.

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