RESUMO
Ribonuclease (RNase)-mediated degradation of messenger RNA (mRNA) poses a huge obstruction to inâ vivo mRNA delivery. Herein, we propose a novel strategy to protect mRNA by structuring mRNA to prevent RNase attack through steric hinderance. Bundling of mRNA strands through hybridization of RNA oligonucleotide linkers allowed the preparation of mRNA nano-assemblies (R-NAs) comprised of 7.7 mRNA strands on average, mostly below 100â nm in diameter. R-NA formation boosted RNase stability by around 100-fold compared to naïve mRNA and preserved translational activity, allowing protein production. A mechanistic analysis suggests that an endogenous mRNA unwinding mechanism triggered by 5'-cap-dependent translation may induce selective R-NA dissociation intracellularly, leading to smooth translation. R-NAs showed efficient mRNA transfection in mouse brain, demonstrating the feasibility for inâ vivo administration.
Assuntos
Nanoestruturas/química , RNA Mensageiro/química , Ribonucleases/metabolismo , Conformação de Ácido Nucleico , Estabilidade de RNA , Ribonucleases/químicaRESUMO
Osmotic stress-induced injured cells of Escherichia coli were prepared by sorting live cells onto tryptic soy agar (TSA) containing 10-50% sucrose. The time course of colony-forming rate (CFR%) was analyzed. A time delay in colony formation indicated a sublethal effect. The final CFR level at 24 h indicated the relative number of culturable cells irrespective of injury. A value of (100-CFR)% at 24 h indicated a lethal effect. When cells were grown on TSA containing 10% sucrose, the time delay was 4 h and the lethal effect was 4%. However, dead cells inhibited the growth of live cells. Physical contact with insoluble matter derived from dead cells or dead cells themselves might have caused growth inhibition. These findings highlight a novel perspective on colony count methods in practical situations, such as when sampling foods containing a high concentration of sucrose.