Impact of Coding Region Accessibility and Uridine Chemistry on Translation in mRNA-DNA Hybrid Origami

Abstract

Messenger RNA (mRNA) is a prerequisite for programmable protein expression, but its therapeutic and synthetic-biology applications are limited by instability and susceptibility to degradation. Hybridizing mRNA to short DNA strands can fold it into a compact origami nanostructure, protecting it from degradation but impeding ribosome access. However, how such a folded mRNA is translated, and which parts must be left unpaired, remain unclear. Here we fold an EGFP-encoding mRNA into a six-helix bundle and leave defined regions of the coding sequence unpaired to examine what the ribosome requires. We find that the start of the coding sequence must be accessible for translation, whereas leaving the far end unpaired makes no difference. Counterintuitively, leaving more of the coding sequence unpaired does not help: translation first falls and then partially recovers as the unpaired region lengthens, a reproducible pattern set by how that region folds rather than by its length. Modified mRNAs carrying 5-methoxyuridine or N1-methylpseudouridine still fold correctly into the six-helix bundle and show the non-monotonic translation pattern; the modification only shifts the overall level of protein produced, with N1-methylpseudouridine giving the most. Together these results begin to define how a folded mRNA can be made both stable and efficiently translated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/734245v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@a54ac6org.highwire.dtl.DTLVardef@1d02202org.highwire.dtl.DTLVardef@168f0cforg.highwire.dtl.DTLVardef@99b51e_HPS_FORMAT_FIGEXP M_FIG C_FIG