Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Abstract

Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5'-coupled conjugate and the other the 3'-coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.

Fig. 1.

Comparison of hypothesized docking conformations between membranes presenting (A) SNARE proteins (adapted from ref. 13) and (B) complementary membrane-anchored DNA in the appropriate orientations. The dimensions of the SNARE complex and a 24-mer hybridized DNA are roughly to scale.

Fig. 2.

Possible stages in DNA-mediated fusion of a pair of lipid vesicles. Separately labeled vesicles present complementary oligomers of DNA coupled to the membrane surface at the 5′- and the 3′-ends. Upon mixing, hybridization of the DNA docks the vesicles together (measured by vesicle co-localization), bringing them into close apposition, followed by a transition to hemifusion that allows mixing of the outer leaflet lipids of the membranes (measured by FRET or FRET dequenching between dye-labeled lipids). Full fusion is achieved when both leaflets merge and contents exchange (measured by observing the onset of fluorescence from Tb(DPA)³⁺ when Tb⁺³ in one vesicle mixes with DPA in another). Note that two DNA-lipid conjugates are shown hybridizing for purposes of illustration, but the actual number required at each step is not known.

Fig. 3.

Graphical illustrations of DNA-lipid conjugates in the 5′ /5′ orientation used to tether vesicles to supported bilayers and allow vesicle docking. (A) Vesicles presenting sequence β are tethered to a supported lipid membrane presenting the complementary sequence β′. These tethered vesicles are observed to diffuse in the plane parallel to the supported membrane. Although they collide, they do not exchange lipids or lose contents (20). (B) When a complementary pair, α and α′, of DNA-lipid conjugates are displayed on separate vesicles, docking is observed upon collisions depending on the number density, sequence and length of the DNA that is displayed (22).

Fig. 4.

Synthetic method for the lipid phosphoramidite for use as the terminal “base” on a DNA-synthesizer (23).

Fig. 5.

Fusion mediated by sequence α vs. T₂₄α. (A) Comparison of the expected docking conformations of vesicles presenting either nonlinker sequences (blue DNA only) and poly T (T_n, shown for n = 24) linker sequences (blue and red DNA). Although the single-stranded linker DNA (red) is drawn as a helix, it is likely to be mostly unstructured; two hybrids are shown purpose of illustration only. Representative kinetic traces for (B) lipid and (C) content mixing collected when two populations of vesicles presenting on average 50 copies of DNA were mixed at equal amounts to a concentration of 2.8 nM in vesicles. Color-coding of the traces is illustrated. Blue: both vesicle populations present only nonlinker sequences 5′-α/3′-α′; Red: both vesicle populations present an average of 50 DNA each (100 total) of nonlinker and linker sequences 5′-α + 5′-T₂₄α/3′-α′ + 3′-T₂₄α′; Green: one vesicle population presents nonlinker sequence 5′-α, and the other presents linker sequence 3′-T₂₄α′; Magenta: both vesicle populations present linker sequences 5′-T₂₄α/ 3′-T₂₄α′; Yellow: vesicles present no DNA (negative control).

Fig. 6.

Effects of linker length on DNA-mediated fusion. (A) Expected conformation when a vesicle presenting nonlinker sequences (blue DNA) reacts with one presenting a T_n linker sequence (blue and red DNA). The single-stranded linker DNA is expected to be unstructured; two hybrids are shown purpose of illustration only. Representative kinetic traces for (B) lipid and (C) content mixing collected when two populations of vesicles presenting an average of 50 copies of DNA were mixed at equal amounts to a concentration of 2.8 nM in vesicles. Color coding of the traces is shown, where the number depicts the length of the oligo-T linker (T_n). In all experiments, the linker sequence 5′-T_nα was included in one population of vesicles and the nonlinker sequence 3′-α′ was included in the other. Blue, n = 0; Red, n = 2; Green, n = 4; Magenta, n = 6; Yellow, n = 12; Cyan, n = 24.