Lipophilic Conjugates for Carrier-Free Delivery of RNA Importable into Human Mitochondria

Abstract

Defects in human mitochondrial genome can cause a wide range of clinical disorders which still do not have efficient therapies. The natural pathway of small non-coding RNA import can be exploited to address into mitochondria therapeutic RNAs. To create an approach of carrier-free targeting of RNA into living human cells, we designed conjugates containing a cholesterol residue and developed the protocols of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable pH-triggered hydrazone bond. The biodegradable conjugates of importable RNA with cholesterol can be internalized by cells in a carrier-free manner; RNA can then be released in the late endosomes due to a change in pH and partially targeted into mitochondria. Here we provide detailed protocols for solid-phase and "in solution" chemical synthesis of oligoribonucleotides conjugated to a cholesterol residue through a hydrazone bond. We describe the optimization of the carrier-free cell transfection with these conjugated RNA molecules and methods for evaluating the cellular and mitochondrial uptake of lipophilic conjugates.

Mitochondrial tRNA-Derived Fragments and Their Contribution to Gene Expression Regulation

“…Import and export through the mitochondrial membrane currently remain a mystery. For this reason, experimental tools, such as specific RNA-aptamers, antisense oligonucleotides, or even delivery of free RNA, could be developed to carefully and reliably determine mt-tRFs spatiotemporal localization (Dovydenko et al, 2021). The putative mechanisms which control import and export of mt-tRNAs and mt-tRFs could be targeted to treat mitochondrial diseases or to ameliorate secondary effects.…”