Structurally reconfigurable designer RNA structures for nanomachines

Abstract

Structurally reconfigurable RNA structures enable dynamic transitions of the functional states in response to diverse molecular stimuli, which are fundamental in genetic and epigenetic regulations. Inspired by nature, rationally designed RNA structures with responsively reconfigurable motifs have been developed to serve as switchable components for building engineered nanomachines, which hold promise in synthetic biological applications. In this review, we summarize recent progress in the design, synthesis, and integration of engineered reconfigurable RNA structures for nanomachines. We highlight recent examples of their targeted applications such as biocomputing and smart theranostics. We also discuss their advantages, challenges as well as possible solutions. We further provide an outlook of their potential in future synthetic biology.

Keywords: Biocomputing; RNA nanomachine; Reconfiguration; Structure; Theranostics.

Figure 1

RNA structures with functions that can be switched via structural reconfigurations. A Ribozymes and aptamers which are structurally responsive to specific molecules. B Riboregulators for trans-acting regulation of gene expression. C Riboswitches for cis-acting regulation of gene expression. D General schematic of CRISPR switches allowing controllable activities of the CRISPR-Cas system

Figure 2

A Schematic of an anti-terminator STAR. This system inverts the structural configuration of the attenuator through the addition of an anti-terminator sequence. Adapted from Meyer et al. . B Pumilio protein-mediated mRNA secondary structural switch controls the accessibility of microRNA-binding sites and regulates the protein expression. RISC, RNA-induced silencing complex. C Translation control of gene expression through a dual protein-dependent RNA secondary structural switch that responds to interferon-γ (IFN-γ). Adapted from Liu et al. (2016a). D Engineered riboregulators responsive to small molecules, which control the gene translation in a trans-acting manner. Adapted from Agapakis and Silver and Munzar et al. . E Toehold switch system comprising a switch RNA for repressing translation, and a trigger RNA with arbitrary sequence which can reconfigure the switch RNA via a toehold-mediated linear-linear interaction. Adapted from Green et al.

Figure 3

A An RNA tetrahedron assembled by the three-way junction (3WJ) motifs from pRNA. Adapted from Li et al. . B Rolling circle transcription (RCT) for the self-assembly of RNA-microsponges. Adapted from Yuan et al. . C RNA origami structure generated via a cotranscriptional folding pathway. The T7 RNA polymerase binds to the template DNA (step 1) and the RNA folds as it is being synthesized (steps 2 to 7). Adapted from Geary et al. . D Molecular design of a triangular RNP assembled based on protein-RNA interactions. Adapted from Durbin et al. . E Design and characterization of an engineered myosin with an RNA lever arm. Adapted from Saper and Hess

Figure 4

A Design of a trigger YES gate for the activation of telomere imaging. Adapted from Hao et al. . B Principle of strand displacement switchable gRNAs. RNA trigger binds the SD gRNA, thereby restoring the gRNA handle. Binding of Cas12a leads to cleavage of the gRNA, and creates an active Cas12a-gRNA complex. Adapted from Oesinghaus and Simme . C Schematic of the activation of theophylline aptazymes modified sgRNA in the presence of theophylline. Adapted from Tang et al. . D Schematic representation of the activation of sgRNA by miRNA triggered cleavage. Adapted from Zhu et al.

Figure 5

A Design schematic for the four-input AND circuit by three toehold switches, two orthogonal transcription factors, and a GFP reporter. Adapted from Green et al. . B Ribocomputing system using RNA molecules as input signals and fluorescent protein as the output signal. Signal processing is carried out by a gate RNA that co-localizes sensing and output modules. Adapted from Simmel et al. . C Design of a 3WJ repressor NAND gate. In the gate RNA, two switch modules are inserted in-frame and upstream of the reporter gene and both input RNAs must bind to the gate to prevent gene expression. Adapted from Kim et al.

Figure 6

A Zika virus toehold switch sensor. The target RNA from the Zika virus can trigger the reconfiguration of the switch RNA and activate the expression of the reporter gene lacZ. Adapted from Pardee et al. . B Schematic of toxin mRNA toehold switch sensor function. Adapted from Takahashi et al. . C Principle of SNIPR for detection of epitranscriptomic marks, which can identify epigenetically modified nucleobases in target RNAs. Adapted from Hong et al. . D An RNP nanomachine for inducing tumor cell apoptosis by oligomerization of apoptosis regulatory proteins. Adapted from Shibata et al.