Lipidated Polyaza Crown Ethers as Membrane Anchors for DNA-Controlled Content Mixing between Liposomes

Philipp M G Löffler,Oliver Ries,Kristian T Sørensen,Stefan Vogel,Alexander Rabe,Ulla Jakobsen,Kasper Glud,Anders Højgaard Hansen

doi:10.1038/s41598-019-49862-y

Philipp M G Löffler, Oliver Ries + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-49862-y

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Abstract

The ability to manipulate and fuse nano-compartmentalized volumes addresses a demand for spatiotemporal control in the field of synthetic biology, for example in the bottom-up construction of (bio)chemical nanoreactors and for the interrogation of enzymatic reactions in confined space. Herein, we mix entrapped sub-attoliter volumes of liposomes (~135 nm diameter) via lipid bilayer fusion, facilitated by the hybridization of membrane-anchored lipidated oligonucleotides. We report on an improved synthesis of the membrane-anchor phosphoramidites that allows for a flexible choice of lipophilic moiety. Lipid-nucleic acid conjugates (LiNAs) with and without triethylene glycol spacers between anchor and the 17 nt binding sequence were synthesized and their fusogenic potential evaluated. A fluorescence-based content mixing assay was employed for kinetic monitoring of fusion of the bulk liposome populations at different temperatures. Data obtained at 50 °C indicated a quantitative conversion of the limiting liposome population into fused liposomes and an unprecedently high initial fusion rate was observed. For most conditions and designs only low leakage during fusion was observed. These results consolidate LiNA-mediated membrane fusion as a robust platform for programming compartmentalized chemical and enzymatic reactions.

Highlights

In recent years, techniques involving Lipid-nucleic acid conjugates (LiNAs) for controlled aggregation[3,4,5,6] and fusion[7,8,9,10] of phospholipid-based liposomes have been developed, but only recently, substantial content mixing (CM) between LiNA-encoded liposome populations has been reported (i.e. ≥20% of encapsulated volume)[11,12]
intensity at time t (It) was estimated that the number of LiNA duplexes per liposome required to open a fusion pore is around 10–209,13, while native sensitive-factor attachment receptors (SNAREs) are effective at 1-2 complexes per vesicle[26]
In our efforts to develop a robust platform for sequence-programmable membrane fusion to be applied in systems biochemistry and as nanoreactors, we have previously studied the effect of different anchor moieties on fusion efficiency, i.e. two linear, branched or planar moieties were attached to the same N-atom of a 3-amino-1,2-propanediol scaffold[34]

Summary

Introduction

Techniques involving LiNAs for controlled aggregation[3,4,5,6] and fusion[7,8,9,10] of phospholipid-based liposomes have been developed, but only recently, substantial content mixing (CM) between LiNA-encoded liposome populations has been reported (i.e. ≥20% of encapsulated volume)[11,12]. SNARE-model systems such as SNARE sub-domains (SNAREpins)[17] and SNARE-derived lipidated coiled-coil peptides[18,19] were shown to induce fusion and content mixing of liposomes. PNAs with aliphatic membrane anchors were employed[23], serving as a useful uncharged alternative to LiNAs. Many of the earlier fusion systems have been reviewed in the literature showing either low levels of content mixing or very substantial leakage for designs with higher levels of content mixing[24,25]. Compared to SNARE-derived lipidated coiled-coil peptides, only a low degree of functionalization of LiNAs is needed to facilitate efficient content mixing between liposomes (~0.5 to 5 mol% versus 0.01 to 0.1 mol% of total lipid for lipopeptides and LiNAs, respectively)[18,19]. It was estimated that the number of LiNA duplexes per liposome required to open a fusion pore is around 10–209,13, while native SNAREs are effective at 1-2 complexes per vesicle[26]

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