| Title: |
Quantitative analysis of oligonucleotide delivery into isolated mitochondria: a proof-of-concept method |
| Authors: |
Joshua McHale; Veronica Bazzani; Abdechakour Elkihel; Jing Wu; Ling Peng; Carlo Vascotto |
| Contributors: |
Mchale, Joshua; Bazzani, Veronica; Elkihel, Abdechakour; Wu, Jing; Peng, Ling; Vascotto, Carlo |
| Publication Year: |
2026 |
| Collection: |
Università degli Studi di Udine: CINECA IRIS |
| Subject Terms: |
Oligonucleotide delivery; mitochondria; subcellular localization; dendrimer; microfiltration |
| Description: |
Mitochondria, with their own DNA, Represent a potential target for nucleic acid-based precision therapies. However, effective delivery of therapeutic oligonucleotides remains challenging due to the dual mitochondrial membranes and the localization of mitochondrial DNA within nucleoid complexes in the matrix. To understand the delivery process and assess the delivery efficiency of potential vectors, such as dendrimers, it is essential to effectively quantify the oligonucleotides that are successfully delivered to and remain within mitochondria. Currently, there are only limited yet inconvenient methods available for this purpose. Here, we describe a method for quantifying the delivery of fluorescent oligonucleotide cargos in isolated mitochondria using a microfiltration apparatus for reliable fluorescent analysis. By working within a range of dilutions, we are able to safeguard the concentration limits. The quantification protocol also enables the visualization of specific localization within mitochondria, allowing for the determination of whether delivery can occur across both membranes. This is particularly useful, as it offers a key insight into improving vectors as they must deliver the cargoes within the mitochondrial matrix. We validate this method in this proof-of-concept study, providing biological data to assess the difference between two amphiphilic dendrimer vectors for oligonucleotide delivery in mitochondria. |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| Relation: |
journal:BIOTECHNIQUES; https://hdl.handle.net/11390/1326305 |
| DOI: |
10.1080/07366205.2026.2635461 |
| Availability: |
https://hdl.handle.net/11390/1326305; https://doi.org/10.1080/07366205.2026.2635461 |
| Rights: |
info:eu-repo/semantics/openAccess ; license:Creative commons ; license uri:http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| Accession Number: |
edsbas.186BE5EC |
| Database: |
BASE |