circSHAPE-MaP for probing the secondary structure of ivcRNAs (ivc-SV-A-cargo).
An internal ribosome entry site (IRES) element is typically required to drive protein translation from in vitro circularized RNAs (ivcRNAs). Here, we evaluate effects of 45 viral IRES elements on ivcRNA translation, and identify that several type V IRESs, including the one from salivirus A (SV-A), efficiently trigger firefly Luciferase translation from ivcRNAs. However, these IRESs display significant translation heterogeneity when engineered with diverse cargo sequences. Structural analyses reveal that alterations in IRES structures within the ivcRNAs critically influence their functionalities. Specifically, the translation-inefficient ivc-SV-A-gE_jcat exhibits an overall disrupted SV-A structure in domain IV due to its extensive interaction with the gE_jcat sequence. Accordingly, unraveling the SV-A-gE_jcat pairing to restore the structural integrity of SV-A domain IV results in efficient gE expression in various cell types. Together, in addition to codon optimization and RNA stability, IRES-cargo interface structurally dictates circular RNA translation, calling attention on the design of translatable ivcRNAs.