ClickIn: a flexible protocol for quantifying mitochondrial uptake of nucleobase derivatives

Abstract

There is an increasing interest in targeting molecules to the mitochondrial matrix. Many proteins are naturally imported through the translocase complexes found in the outer and inner mitochondrial membranes. One possible means for importing molecules is therefore to use a mitochondrial pre-protein as a vector and assess what forms of molecules can be attached to the pre-protein as cargo. A major difficulty with this approach is to ensure that any chimaeric molecule does indeed access the mitochondrial matrix and does not merely associate with the mitochondrial membranes. We have recently demonstrated that click chemistry can be used both to demonstrate convincingly mitochondrial import of a peptide–peptide nucleic acid conjugate and also to quantify the mitochondrial uptake for specific synthetic conjugates. We now report an adaptation of the synthesis to facilitate simple quantification of multiple molecules and hence to calculate the efficiency of their mitochondrial import.

Advances in methods for reducing mitochondrial DNA disease by replacing or manipulating the mitochondrial genome

“…In addition, doubts arose concerning the reliability of current methods to confirm and detect uptake into the mitochondrial matrix [ 42 ]. To circumvent these issues, a new methodology named “ClickIn” was developed [ 43 , 44 ]. In this method, MitoOct, a bio-orthogonally reactive TPP accumulates in the mitochondrial matrix of energized mitochondria, where it will react at an accelerated rate with azide labelled PNA, therefore selectively labelling matrix localized PNA.…”

Signed‐For Delivery in the Mitochondrial Matrix: Confirming Uptake into Mitochondria

“…C) UV‐crosslinking and immunoprecipitation (CLIP) can covalently bind imported proteins to RNA species occurring only in the mitochondrial matrix, immunoprecipitation of the protein and sequencing of the crosslinked RNA can confirm uptake in the mitochondrial matrix . D) ClickIn: a bioorthogonal reaction between an azide‐labeled peptide or peptide nucleic acid and a mitochondria targeted cyclooctyne (MitoOct) can be used to detect uptake in the mitochondrial matrix …”

“…This locally increased concentration accelerates the click reaction, and the resulting yield over time can be interpreted as a measure of the mitochondrial membrane potential . Hoogewijs et al adapted this technique to develop the ClickIn method, in which a mitochondrially targeted peptide or peptide nucleic acid is labeled with azido‐lysine at the C‐terminus and incubated with the cyclooctyne MitoOct in the presence of isolated mitochondria (Figure D) . Once the peptide has been taken up in to the matrix and passed through the TIM, its azide tag is exposed to a high concentration of MitoOct accumulated there by the membrane potential, yielding a significantly higher amount of crosslinked or “clicked” product compared to uncoupled mitochondria in which MitoOct does not accumulate.…”

Chemical biology of mitochondria

“…From this general introduction, the following articles focus on particular aspects of the chemical biology of mitochondria. In the article by Hoogewijs et al [2], 'Click Chemistry' is used to confirm whether or not a construct has made it successfully to the mitochondrial matrix, opening up the way to assessing the uptake of a range of constructs in the future.…”