d, Dicistroviridae family IRES structure including three loops critical for translational activity and insertion sites. c, Activities of different IRES modules in HEK293T cells with and without co-transfection with T7 RNAP and GFP trigger sequence. Plasmids encoding IRES or eToehold candidates upstream of a reporter protein, polymerase (for example, T7 RNAP) and trRNA sequence (for example, GFP) were co-transfected into HEK293T cells. b, Basic screening methodology for eToeholds. trRNA activates IRES activity through strand invasion and release of the IRES into an activated state, allowing for ribosome binding and protein production. We also demonstrate the capability of eToeholds to discriminate different cell states and cell types by selectively activating protein translation based on endogenous RNA levels.Ī, eToehold modules are in a locked state in which IRES activity is inhibited, preventing ribosome recruitment and translation. We further show that stable cell lines expressing eToeholds can be used to sense natural viral infection (by Zika virus) and viral transcripts (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) constructs). We demonstrate eToehold functionality in human and yeast cells as well as cell-free lysates. Using this system, we achieve up to 16-fold trRNA-induced translation of transgenes. These eToeholds incorporate modified IRESs that are designed to be inactive until sense–antisense interactions with specific trRNAs cause activation (Fig. In this study, we developed RNA-based eukaryotic modules, called eToeholds, that enable the regulated translation of in cis reporter genes by the presence of specific trRNAs. Internal ribosome entry sites (IRESs) are RNA elements found in several viruses and endogenous eukaryotic transcripts whose structure has evolved to initiate protein translation independent of mRNA 5′ capping and polyadenylation. Although recent studies have used ribozymes 8, 9 to respond to short nucleotide oligomers, these ribozyme-based sensors are not yet compatible with the detection of longer trRNAs, including endogenous transcripts. A more robust technique involves the use of a ribozyme that cleaves the polyA tail upon small molecule induction 6, 7. However, these mechanisms induce only modest fold changes in both eukaryotes and prokaryotes. Unfolding of the gRNA by trRNA leads to activation of the Cas9 enzyme and corresponding downstream regulation. More complex RNA-based switches have been developed, using Cas9 expression and engineered folding of the guide RNA (gRNA) to hide sequences essential for function 4, 5. The 5′ cap dominates translational regulation mechanisms and is a major challenge for any eukaryotic RNA-sensing riboswitch that functions at the level of translation. Although the Kozak sequence improves ribosomal binding, it is not as critical to translation as the prokaryotic RBS previously developed Kozak-based toehold switches have achieved only up to two-fold trRNA-driven induction of eukaryotic translation 3. Eukaryotic translation, however, is far more complicated and is typically regulated by several factors, including 5′ modified capping recruited by RNA polymerase II, a poly-adenosine (polyA) tail for mRNA stabilization and a Kozak consensus sequence for protein translational regulation. The RBS is released when a trRNA binds the toehold switch and opens the stem loop structure, thus initiating translation of the reporter gene. Toehold switches selectively repress translation of an in cis reporter gene by sequestering the ribosome binding site (RBS) upstream of the reporter gene in a stem loop structure in the absence of a trRNA. We previously developed RNA-based prokaryotic modules, called toehold switches, for detecting specific RNA transcripts 1, 2. Synthetic biology techniques for sensing and responding to specific intracellular RNAs are desirable for therapeutic and diagnostic applications, as they provide a means to discriminate and target specific cells, tissues and organisms and can serve as building blocks for sophisticated genetic circuits.
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