Bivalent Cholesterol-Based Coupling of Oligonucletides to Lipid Membrane Assemblies

Rapid progress in protein-chip technologies is today made with respect to water soluble proteins, but to generate a signature of the whole proteome makeup, membrane proteins, which constitute an important group of proteins being a common target in disease diagnostics and therapeutic drugs, must also be addressable. This class of proteins is often identified as an extremely difficult group of proteins to be analyzed on this format. In fact, the first lowdensity protein chip based on membrane proteins was only recently reported, demonstrating an array produced via microdispensing of G protein-coupled receptor containing lipid membranes. However, to fully explore the potential of array-based analysis of membrane proteins, tethered lipid Vesicles have recently emerged as a most promising alternative, not the least since they offer the possibility to measure also membrane-protein-mediated material transport across the membrane. Nevertheless, efficient means to control the positioning of different types of vesicles on predefined regions are still lacking. By combining the concept of DNA-labeled vesicles, previously utilized for signal enhancement of DNA hybridization detection, with the concept of using DNA-labeled biomolecules for site-selective binding on cDNA arrays, we and others recently demonstrated the use of cDNA arrays for siteselective and sequence-specific coupling of DNA-tagged lipidvesicles. Instead of using covalent coupling of DNA to chemically active lipids, we made use of cholesterol-modified ss-DNA for spontaneous anchoring into the hydrophobic interior of lipid membranes. This means of anchoring DNA adds a threefold advantage: the method (i) is faster, (ii) does not require chemically modified lipids to be introduced, and (iii) makes use of a naturally occurring membrane constituent, thus eliminating the risk for side effects induced by chemically reactive lipid headgroups on incorporated membrane constituents. However, the cholesterol-based anchoring of DNA turns out to be relatively weak, thus complicating quantitative control of the number of DNA per vesicles. In addition, site-selective sorting of different DNA-tagged vesicles to cDNA arrays must, because of influence from DNA exchange between differently tagged vesicles, be accomplished in a sequential, rather than parallel, manner.