Targeted cell imaging properties of a deep red luminescent iridium(iii) complex conjugated with a c-Myc signal peptide.

Adam H Day,Emyr Lloyd-Evans,Robert J Mart,Rudolf K Allemann,Ian A Fallis,Niklaas J Buurma,Martin H Übler,Nathaniel I Keymer,Hannah L Best,Eman A H Al-Wattar,Simon J A Pope

doi:10.1039/c9sc05568a

Adam H Day, Emyr Lloyd-Evans + Show 9 more

Open Access

https://doi.org/10.1039/c9sc05568a

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Abstract

A nuclear localisation sequence (NLS) peptide, PAAKRVKLD, derived from the human c-Myc regulator gene, has been functionalised with a long wavelength (λ ex = 550 nm; λ em = 677 nm) cyclometalated organometallic iridium(iii) complex to give the conjugate Ir-CMYC. Confocal fluorescence microscopy studies on human fibroblast cells imaged after 18-24 h incubation show that Ir-CMYC concentrations of 80-100 μM promote good cell uptake and nuclear localisation, which was confirmed though co-localisation studies using Hoechst 33342. In comparison, a structurally related, photophysically analogous iridium(iii) complex lacking the peptide sequence, Ir-PYR, showed very different biological behaviour, with no evidence of nuclear, lysosomal or autophagic vesicle localisation and significantly increased toxicity to the cells at concentrations >10 μM that induced mitochondrial dysfunction. Supporting UV-visible and circular dichroism spectroscopic studies show that Ir-PYR and Ir-CMYC display similarly low affinities for DNA (ca. 103 M-1), consistent with electrostatic binding. Therefore the translocation and nuclear uptake properties of Ir-CMYC are attributed to the presence of the PAAKRVKLD nuclear localisation sequence in this complex.

Highlights

A signal peptide is a short peptide sequence o en present at the N-terminus of newly synthesised proteins that is responsible for intra- and extracellular translocation.[1]
We were interested in an alternative nuclear localisation sequence (NLS) peptide sequence (PAAKRVKLD) which is derived from the human c-Myc protein and was rst identi ed in 1988 to be essential for nuclear localisation of the protein.[5]
We wished to visualise the cellular translocalisation characteristics of the c-Myc NLS (PAAKRVKLD) in human broblast cells. To do this we investigated the use of a deep red luminescent organometallic complex as an optical label for the c-Myc NLS, which we envisaged would be advantageous when applied to confocal uorescence microscopy

Summary

Introduction

A signal peptide is a short peptide sequence o en present at the N-terminus of newly synthesised proteins that is responsible for intra- and extracellular translocation.[1]. Targeted cell imaging properties of a deep red luminescent iridium(III) complex conjugated with a c-Myc signal peptide† A nuclear localisation sequence (NLS) peptide, PAAKRVKLD, derived from the human c-Myc regulator gene, has been functionalised with a long wavelength (lex 1⁄4 550 nm; lem 1⁄4 677 nm) cyclometalated organometallic iridium(III) complex to give the conjugate Ir-CMYC.

Results

Conclusion