Inspired by a previously described strategy for vesicle tethering , we designed an experimental configuration where it is possible to increase the likelihood for viruses to bind close to the equatorial regions of the target vesicles and achieve fusion in a side-by-side configuration. We prepared a supported lipid bilayer (SLB) using the gel-phase (at room temperature) forming lipid DPPC and small fractions of two sets of orthogonal DNA-lipid conjugates (L-dN241 and L-dN242, Figure ) that can serve as binding sites for objects (vesicles and viruses) bearing DNA-lipid conjugates with complementary sequences (i.e., L-dN241′ and L-dN242′, respectively). We chose a mole fraction of the DNA-lipid conjugates with respect to DPPC such that there are approximately 4 DNA-lipid conjugates of each type on average within a 100 nm×100 nm region of the SLB. We assumed that some virus particles incorporating a DNA-lipid conjugate will initially dock at pH 7.4 on to vesicles via GD1a receptors, following which they will also tether strongly to the SLB by hybridizing with a nearby complementary DNA sequence. In this way, a vesicle-virus pair can be positioned next to each other on the SLB and, because of the gel nature of the SLB, they will not be free to diffuse, essential for the super-localization measurements described in the following. Also, the gel-phase membranes are expected to be mechanically stiffer than fluid phase membranes and therefore the virions are not expected to fuse to the SLB itself . We first tethered target vesicles displaying GD1a to the SLB via DNA-lipid conjugate L-dN241′ (Figure ) and washed away unbound vesicles (Figure ). In control conditions where no DNA-lipid conjugate was added to the SLB or the target vesicles or both, no or minimal tethering of vesicles to the SLBs was detected. Next, we added the virus particles pre-incubated with DNA-lipid conjugate L-dN242′ and allowed them to simultaneously attach to GD1a receptors on the vesicle surface and to the DNA strand on the SLB surface. We noted that attachment of a virus particle next to a vesicle is an inherently low probability event. Only 10 virus particles (internally labeled with QuantiFluor) per 1000 target vesicles were observed to tether to the SLB via DNA-lipid conjugates next to a vesicle when the vesicles lacked GD1a. However, when GD1a was present on the vesicles, the probability of association increased ~4 times (43 per 1000) (Figure ).