Tailoring Interleaflet Lipid Transfer with a DNA-based Synthetic Enzyme

Abstract

Lipid membranes, enveloping all living systems, are of crucial importance, and control over their structure and composition is a highly desirable functionality of artificial structures. However, the rational design of protein-inspired systems is still challenging. Here we have developed a highly functional nucleic acid construct that self-assembles and inserts into membranes, enabling lipid transfer between inner and outer leaflets. By designing the structure to account for interactions between the DNA, its hydrophobic modifications, and the lipids, we successfully exerted control over the rate of interleaflet lipid transfer induced by our DNA-based enzyme. Furthermore, we can regulate the level of lipid transfer by altering the concentration of divalent ions, similar to stimuli-responsive lipid-flipping proteins.

Cations Regulate Membrane Attachment and Functionality of DNA Nanostructures

“…To this end, we consider the functionality of 2C DNA constructs, which upon membrane insertion form toroidal pores in lipid bilayers, triggering the exchange of lipids between the inner and outer leaflets, similar to scramblase enzymes. 12 , 13 Here we report that the activity of such a synthetic enzyme can be triggered with cations, remarkably alike the natural scramblases. 66 …”

“…The construct we first consider in this section is a DNA duplex ( Figure 3 a and Table S1 ) tagged with two cholesterol molecules (2C) as reported previously. 13 Its ability to decorate the surface of POPC GUVs (L d phase) was studied at room temperature for a range of magnesium concentrations between 0 and 4 mM, spanning the physiologically relevant values for serum (0.75–1.25 mM 61 ). The stability of the construct was not significantly affected by changes in cation concentration, as shown with PAGE ( Figure S14 ) and UV–vis absorbance spectrophotometry ( Figure S15 ).…”

“…In addition, the understanding of DNA–lipid interactions underpins our ability to engineer and interface lipid membranes with synthetic DNA nanostructures, designed to replicate key functionalities of biological membrane proteins, including the ability to remodel bilayers, 9 − 11 induce lipid flipping 12 , 13 and regulate ion transport, 14 − 18 membrane–membrane adhesion, 19 , 20 and fusion. 21 − 23 …”

“… 26 − 29 Accounting for the effect of (physiological) ions is therefore critical to inform the design of both NA–lipid formulations and membrane-active DNA nanodevices. The latter, in particular, often feature complex and programmable morphologies, charge distributions, and chemical modifications, 9 , 13 , 30 whose coupling with the electrostatic effects mediated by cations could unlock novel functionalities.…”

Membrane activity of a DNA-based ion channel depends on the stability of its double-stranded structure

“…Cholesterol-modified DNA will attach to a membrane, but only when it is pulled with opposing cholesterol molecules will DNA insert into the bilayer. Building of biological nucleic acid nanopores is thus based on designing such architectures, with hydrophobicity distributed in a way that forces the hydrophilic DNA to span the membrane 10,19,22,24 . Aiming at finding a comprehensive description of design principles, numerous variables are tested, including shape 24,25 , number and position of hydrophobic modifications 10,13,26 and cholesterolmediated clustering 27 .…”