Opportunities in the design and application of RNA for gene expression control

Abstract

The past decade of synthetic biology research has witnessed numerous advances in the development of tools and frameworks for the design and characterization of biological systems. Researchers have focused on the use of RNA for gene expression control due to its versatility in sensing molecular ligands and the relative ease by which RNA can be modeled and designed compared to proteins. We review the recent progress in the field with respect to RNA-based genetic devices that are controlled through small molecule and protein interactions. We discuss new approaches for generating and characterizing these devices and their underlying components. We also highlight immediate challenges, future directions and recent applications of synthetic RNA devices in engineered biological systems.

Figure 1.

The introduction of high-throughput sequencing (HTS), or next-generation sequencing (NGS), into the SELEX process has revolutionized the selection of new aptamers. The Systematic Evolution of Ligands by EXponential enrichment (SELEX) process selects aptamers de novo from a large sequence library. SELEX can be performed using conventional selection methods or with novel selection approaches including microfluidic SELEX, semi-automated SELEX, CE-SELEX, capture-SELEX and branched SELEX. The enriched pool of potential aptamers is sequenced using NGS and analyzed using a variety of bioinformatic programs and computational methods. Using tools such as FASTAptamer and AptaTools, researchers can analyze the sequencing reads by visualizing the aptamer sequence landscape, identifying motifs from sequence distributions and generating clusters of sequence families.

Figure 2.

Strategies for constructing RNA devices based on higher-ordered structural interactions. (A) The aptamer (red sequence) is coupled to a non-coding RNA (ncRNA). In the absence of ligand (red pentagon), the structural interaction between the aptamer and the ncRNA (blue sequence) inactivates the ncRNA regulatory function. Ligand binding to the aptamer abolishes such structural interactions and activates the ncRNA for translational inhibition. (B) The aptamer (blue sequence) is integrated into the basal segment domain of a microRNA (miRNA). Processing of the miRNA results in incorporation of the targeting strand (red sequence) into the RNA-induced silencing complex (RISC), which silences target gene expression. Ligand (blue hexagon) binding to the aptamer inhibits proper processing of the miRNA, thereby reducing RNAi-mediated gene silencing and increasing target gene expression. (C) The aptamer (purple sequence) is integrated onto one of the loops of a HHRz, which is encoded into the 3′ UTR of a gene. Self-cleavage (at red arrow) destabilizes the transcript for degradation by ribonucleases, hence decreasing gene expression. Ligand (purple circle) binding to the aptamer disrupts tertiary interactions required for self-cleavage, thereby permitting translation of the target gene.

Figure 3.

Applications of RNA devices. (A) In vivo quantification. A miRNA-based device that silences gene expression of a reporter (i.e. GFP) may be used for direct, non-invasive quantification of an intracellular protein (blue oval) or metabolite. (B) Metabolic pathway engineering. A ribozyme-based device encoded in the 3′ UTR of a suicide gene may be used to construct a suicide riboswitch for growth-based selection in yeast. Growth is rescued in the presence of the metabolite (blue diamond), and large enzyme libraries may be screened for variants that produce high titers of metabolite. (C) Programming mammalian cell behavior. Riboswitches may be inserted into viral vectors in the 5′ and 3′ UTRs of a gene of interest (GOI) to externally regulate transgene expression following viral transduction.