Translocation of 5′ mRNA cap analogue — peptide conjugates across the membranes of giant unilamellar vesicles

Abstract

Cell-penetrating peptides (CPPs) have been extensively studied because of their ability to deliver various cargo molecules, which are often potential therapeutic agents. However, in most cases, the exact entry mechanism of CPPs is still unknown. In this study, we focused our attention on the membrane permeability sequence (MPS) peptide (AAVALLPAVLLALLAK) conjugated to analogues of a 5' mRNA cap. This unique RNA structure plays a pivotal role in eukaryotic gene expression and has a large therapeutic application potential. We validated the translocation abilities of conjugates across the membranes of giant unilamellar vesicles (GUVs) composed of POPC lipids by application of fluorescence microscopy. Translocation of the MPS peptide itself was observed in contrast to peptide conjugates containing mono- and dinucleotide cap analogues, indicating that even for such small cargos, passive translocation does not occur. However, membrane permeability was observed in the case of conjugated mononucleotides. Fluorescence lifetime microscopy (FLIM) of the C6-NBD-phospholipid revealed changes in lipid packing induced by a penetrating peptide. Our results support the usefulness of artificial membrane systems applied to elucidate membrane crossing mechanisms.

Effect of HIV-1 TAT Peptide Fusion on 5′ mRNA Cap Analogs Cell Membrane Permeability and Translation Inhibition

“…This indicates that TAT translocation is related to membrane disordering, which is also present to a lesser extent for nontranslocating TAT conjugates. Similar results were collected in our previous studies on MPS-conjugated nucleotides …”

“…The method described by Wheaten et al . allows for assessment of translocation of compounds which are not fluorescently labeled and was used previously in our studies of MPS conjugates . It is based on GUVs incubation with the unlabeled compound of interest and the membrane impermeable fluorescent dye carboxyfluorescein (CF).…”

“…N2-alkyne modified m 7 GMP was synthesized using pent-4-yn-1-amine and identified as described previously. , m 7 GpppG with the second guanosine bearing N2-alkyne modification was obtained and identified as described in detail previously . Coupling reaction of N2-pentynyl m 7 GMP (4) with N 3 -TAT was carried out as folows: 5′-monophosphate N 2-(pent-4-yn-1-yl)-7-methylguanosine (TEA salt) (5 mg, 0.0075 mmol) and N 3 -TAT (12 mg, 0.0075 mmol) were dissolved in DMF (200 μL) with addition of few drops of water under an argon atmosphere.…”

“…POPC GUVs were prepared as described previously . In short, 10 μL of 1 mg/mL POPC in CHCl 3 was applied on two Pt wire electrodes in homemade Teflon chambers.…”

“…The method described by Wheaten et al 17 allows for assessment of translocation of compounds which are not fluorescently labeled and was used previously in our studies of MPS conjugates. 18 It is based on GUVs incubation with the unlabeled compound of interest and the membrane impermeable fluorescent dye carboxyfluorescein (CF). When compounds translocate and form pores, the liposomes and smaller inner vesicles were usually found in a fraction of electroformed GUVs fill with dye.…”