Ribonuclease (RNase)-mediated degradation of messenger RNA( mRNA) poses ah uge obstruction to in vivo mRNAdelivery.Herein, we propose anovel strategy to protect mRNAbystructuring mRNAtoprevent RNase attackthrough steric hinderance.Bundling of mRNAstrands through hybridization of RNAo ligonucleotide linkers allowed the preparation of mRNAn ano-assemblies (R-NAs) comprised of 7.7 mRNAs trands on average,m ostly below1 00 nm in diameter. R-NAf ormation boosted RNase stability by around 100-fold compared to naïve mRNAand preserved translational activity, allowing protein production. Am echanistic analysis suggests that an endogenous mRNAunwinding mechanism triggered by 5'-cap-dependent translation mayi nduce selective R-NAd issociation intracellularly,leading to smooth translation. R-NAs showed efficient mRNAt ransfection in mouse brain, demonstrating the feasibility for in vivo administration.
Invitro-transcribedmRNAisapromisingtherapeuticoptionfor various diseases. [1] However,t he susceptibility of mRNA to ribonuclease (RNase)-mediated degradation in physiological environments is am ajor obstacle to the widespread in vivo application of mRNA. Approaches that have been taken to overcome this issue include the complexation of mRNAw ith cationic polymers,p eptides or lipids, [2] and the chemical modification of mRNAnucleosides. [3] Nevertheless, mRNAd egradation still poses ag reat challenge to the delivery of mRNAt otarget tissues in an intact form.Herein, we tackled this issue by proposing anovel strategy for protecting mRNAb ased on the formation of highly structured mRNAn ano-assemblies (R-NAs). Studies in the growing field of RNAn anotechnology have shown that structured RNApresents enhanced RNase stability probably through steric hinderance to RNase attack, providing an effective delivery platform, for example,f or small interference RNA(siRNA) delivery. [4] However,itis not trivial to use this approach for mRNAdelivery.While the precise design of RNAs equence is required to provide intended base-pairing in the preparation of structured RNA, sequence flexibility is extremely low in mRNAc ompared to that of other applications of RNAn anotechnology.F urthermore,m RNAh as af ixed structure containing a5 '-cap,5 '-a nd 3'-untranslated regions,acoding region, and ap oly-A tail, and all these components play ac ritical role in protein translation and intracellular mRNAm etabolism. [1] Therefore,t op repare structured mRNA, possessing all of these endogenous mRNA components without sequence optimization, we bundled mRNAs trands by hybridizing RNAo ligonucleotide linkers to prepare R-NAs (Figure 1). This simple approach of mRNA engineering boosted the RNase stability of mRNAbyaround 100-fold. Notably,t he translational activity of R-NAs was maintained at al evel comparable to naïve mRNA, presumably due to selective R-NAd issociation in the intracellular environment through an endogenous mRNAu nwinding mechanism triggered by 5'-cap-dependent translation. As aresult, R-NAs achieved efficient mRNAtransfection to the mous...