Synthesis, Incorporation, and Translation Properties of Threose Nucleic Acid Modified Trinucleotide Cap Analogs

Enhancing protein translation remains a key objective in the continued development of mRNA-based therapeutics. One promising strategy involves the chemical modification of the 5' mRNA cap that is essential for RNA stability and translation initiation via eIF4E binding. Given its smaller size, nuclease stability, and unique 3' → 2' internucleotide linkage, we evaluated α-l-threofuranosyl nucleic acid (TNA) modification at the 5'-end of mRNA. TNA modified trinucleotide caps were synthesized and incorporated cotranscriptionally into several mRNAs using T7 polymerase with comparable full-length integrity, yield, and capping efficiency relative to reference cap <b>m7G-A</b> _<b>OMe</b> <b>G</b>. Capped mRNAs incorporating TNA modification were shown to support translation in cell extract and cultured cells. Placement of TNA at the inverted m7G (<b>m7G</b> _<b>TNA</b> <b>-A</b> _<b>OMe</b> <b>G</b>) negatively impacted protein translation relative to the predominant natural Cap 1 structure, but notably, TNA modification at the first transcribed nucleotide improved protein production in all evaluated systems. Biochemical experiments suggest that both TNA modified caps improve decapping resistance <i>in vitro</i> and do not significantly increase proinflammatory interleukin-6 release in HeLa cells. In addition, molecular modeling simulations revealed that caps bearing TNA are well accommodated within eIF4E. Lastly, we show that SpCas9 mRNAs containing TNA cap <b>m7G-A</b> _<b>TNA</b> <b>G</b> cotransfected with single-guide RNA targeting murine transthyretin (<i>mTtr</i>) resulted in efficient gene editing in cultured cells. These findings contribute to the expanding toolkit of modified cap analogs and further our understanding of the chemical tolerability of specific positions within the cap scaffold.