Specific DNA Duplex Formation at an Artificial Lipid Bilayer: towards a New DNA Biosensor Technology

Abstract

A novel technique is described which comprises a base-specific DNA duplex formation at a lipid bilayer-H(2) O-phase boundary layer. Two different probes of oligonucleotides both carrying a double-tailed lipid at the 5'-terminus were incorporated into stable artificial lipid bilayers separating two compartments (cis/trans-channel) of an optically transparent microfluidic sample carrier with perfusion capabilities. Both the cis- and trans-channels are filled with saline buffer. Injection of a cyanine-5-labeled target DNA sequence, which is complementary to only one of the oligonucleotide probes, into the cis-channel, followed by a thorough perfusion, leads to an immobilization of the labeled complementary oligonucleotide on the membrane as detected by single-molecule fluorescence spectroscopy and microscopy. In the case of fluorescent but non-complementary DNA sequences, no immobilized fluorescent oligonucleotide duplex could be detected on the membrane. This clearly verifies a specific duplex formation at the membrane interface.

Specific DNA duplex formation at an artificial lipid bilayer: fluorescence microscopy after Sybr Green I staining

“…Experiment A: 4 + 5 + SG . In a preceeding manuscript we have studied the bilayer incorporation of the lipo-oligonucleotide 4 (5’-d( 1a -TAG GTC AAT ACT)-3’) and its successful and specific duplex formation with the cyanine-5-labeled complementary strand 5 (5’-d[(Cy5)-p-AGT ATT GAC CTA]-3’) [ 19 ]. As a negative control it had been shown that a membrane-bound duplex formation between the lipo-oligonucleotide 7 (5’-d( 1a -ATC CAG TTA TGA)-3’) and the oligomer 5 failed.…”

“…In the first experiment (A) laser irradiation of the intercalated dye was performed at 470 nm. In orienting experiments Sybr Green I was irradiated at 470 nm, in order to see if this dye stains the complementary DNA strands 4 + 5 , which are known to form a duplex at the bilayer membrane [ 19 ]. Firstly, we evaluated the optimal measuring conditions such as the volume, the Sybr Green I concentration as well as of the lipo-oligonucleotides.…”

“…Fig. 5 demonstrates the difference in the bilayer brightness upon irradiation of either cyanine-5 (635 nm) [ 19 ] or of the ternary complex with SG ( 4 + 5 + SG ) (470 nm). For the determination of the bilayer brightness a region-of-interest (ROI) was defined around the bilayer, and subsequently the density of the intensity counts were summarized (for details, see Experimental).…”

“…In a preceding manuscript [ 19 ] we reported the lipid bilayer immobilization of lipo-oligonucleotides carrying a racemic bis(hexadecyloxy)propan-1-yl tag ( 1a ) at the 5’-termini at an artificial lipid bilayer–water phase boundary. These were prepared using the cyanoethyl phosphoramidite 1b .…”

Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms

“…Lipids are commonly used for modifying oligonucleotides. 10 The importance of such hybrid conjugates has been demonstrated in areas like nanobiotechnology, 11 cell biology 12 and medicine. 5 From a therapeutic point of view, lipid-oligonucleotide conjugates (LOCs) have emerged as an alternative to cationic particles in order to reduce toxicity and improve biodistribution.…”