In situ vesicle formation by native chemical ligation

Abstract

Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non-enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water-soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self-assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction makes in situ membrane formation compatible with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes.

Keywords: membranes; native chemical ligation; phospholipids; self-assembly; synthetic biology.

Figure 1

Synthesis of phospholipids by NCL reaction of a cysteine-functionalized lysolipid and MESNA oleoyl thioester. Framed: the mechanism of the NCL and the possible substrate reloading.

Figure 2

Characterization of the amidophospholipid vesicular structure. a) Fluorescence microscopy image of membrane-containing vesicles formed by hydration of a thin film of phospholipid 3. Membranes were stained using 1 μM Texas Red® DHPE dye solution. b) TEM image of negatively stained self-assembled vesicular structures formed by the amidophospholipid 3. c) Fluorescence microscopy image demonstrating the encapsulation of HPTS in phospholipid 3 membrane vesicles. d) Steady-state anisotropy of DPH in membranes formed from amidophospholipids 3 and 4 compared with those from native phosphatidylcholines with the indicated acyl chains. The unitless anisotropy ratio (R) is a measure of the acyl packing of the bilayer, with higher values indicating a more ordered membrane.

Figure 3

In situ phospholipid membrane assembly driven by a non-enzymatic reaction. a) Model of spontaneous vesicle assembly induced by NCL-based amidophospholipid synthesis. b) Fluorescence microscopy image of the membrane-containing vesicles formed by spontaneous assembly directed by NCL-based phospholipid 3 synthesis. Membranes were stained using 1 μM Texas Red® DHPE dye solution. c) Fluorescence microscopy image demonstrating the encapsulation of GFP in vesicles driven by the in situ self-assembly of phospholipid 3 membranes.

Figure 4

In situ vesicle formation (top) and growth (bottom). An aqueous buffer solution of cysteine-functionalized lysolipid 1b (1 mM) and MESNA oleoyl thioester 2 (1 mM) in the presence of TCEP.HCl (20 mM) imaged at different times after initial mixing. The upper panels show phase-contrast images corresponding to the vesicle formation. Initially, phospholipid membranes were not present. However, shortly after mixing of both precursors, the spontaneous formation and growth of vesicle and tubular structures was observed. After 30 min, starting materials were consumed and replaced with large fields of vesicles. The bottom panels are phase contrast micrographs of vesicles growing over a period of 10 minutes in the presence of reactive lipid precursors.