C. D'Amico, M. Mykkanen, S. Saarinen, V. Sakkinen, M. A. Kostiainen
Folded an EGFP-encoding mRNA into a six-helix bundle and varied which parts of the open reading frame were left unpaired, measuring output both within the folded series and against the unhybridized transcript. Exposing the first 35 coding nucleotides raises translation, exposing the last 35 does not; across overhangs of 35 to 100 nucleotides output falls in two discrete steps, with no difference between 50, 60, 70 and 80 nucleotides. A construct with the entire open reading frame paired still translates at two-thirds of the reference, implying that elongating ribosomes displace staples as they read.
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 a mRNA with short DNA staples can fold it into a defined nanostructure and shield it from nuclease degradation, but the same base pairing hides the transcript from the ribosome. Which regions must remain accessible, and how much coding sequence can be given over to structure, is not known. We folded an EGFP-encoding mRNA into a six-helix bundle and varied which parts of the open reading frame were left unpaired, measuring output both within the folded series and against the unhybridized transcript. The requirement is positional and asymmetric: exposing the first 35 coding nucleotides raises translation, exposing the last 35 does not. More exposure is not better: across overhangs of 35 to 100 nucleotides output falls in two discrete steps, with no difference between 50, 60, 70 and 80 nucleotides, indicating a threshold set by the ribosomal initiation footprint rather than a graded dependence on length. A construct with the entire open reading frame paired still translates at two-thirds of the reference, implying that elongating ribosomes displace staples as they read and that the folded state is transient during translation. Importantly, one staple set folds scaffolds built from uridine, 5-methoxyuridine or N1-methylpseudouridine, and a 35-nucleotide overhang is optimal in all three. N1-methylpseudouridine gives the highest output but is threefold more sensitive to an over-long overhang, consistent with its stabilization of RNA secondary structure. Coding-overhang accessibility and uridine chemistry are therefore separable design parameters. Together these results begin to define how a folded mRNA can be made both stable and efficiently translated.