G25 spin column

Prior to analysis by fluorescence recovery after photobleaching
(FRAP), OMVs must be fluorescently labeled. This was achieved by adding
1 μL of 0.36 mM R18 dye (Invitrogen) to 200 μL of OMV
stock and sonicating for 15 min. A G25 spin column (GE healthcare)
was used to remove unbound/excess R18 by centrifugation at 1500 g for 3 min at room temperature. Lipid bilayers were formed
using the protocol outlined above. FRAP measurements were conducted
using an inverted Zeiss LSM800 confocal microscope with a 10×
objective lens. A 30 μm diameter bleaching spot was made, and
recovery of the fluorescence intensity of this spot was measured over
time relative to a 50 μm diameter reference spot. The data were
analyzed using MATLAB, and the fluorescence recovery was modeled using
a modified Bessel function as previously described.^{26 (link)} The model fit was used to extract the diffusion coefficient
(D) according to the equation D = r²/4τ where r is the radius
of the photobleached spot and τ is the characteristic diffusion
time. The fit was also used to extract the mobile fraction (MF) according
to the equation (I_E – I₀)/(I_I – I₀), where I_E is the final
postbleach intensity value, I₀ is the
first postbleach intensity value, and I_I is the initial prebleach intensity value.

Prior to analysis
by FRAP, samples were fluorescently labeled. This was achieved by
adding 1 μL of octadecyl rhodamine chloride 18 dye (R18) (Invitrogen)
to 200 μL of proteoliposome suspension and sonicating for 15
min. A G25 spin column (GE Healthcare) was used to remove unbound/excess
R18 by centrifugation at 3000 rpm for 3 min at room temperature. Lipid
bilayer formation was then conducted using the protocol outlined above.
FRAP measurements were conducted using an inverted Zeiss LSM800 confocal
microscope with a 10× objective lens. A 30 μm diameter
bleaching spot was made, and recovery of the fluorescence intensity
of this spot was measured over time relative to a 50 μm diameter
reference spot. The data were analyzed using MATLAB using the Soumpasis
fit to extract the diffusion coefficient (D) according
to the eq D = r²/4τ
where r is the radius of the photobleached spot and
τ is the characteristic diffusion time. FRAP was performed on
SLBs on glass and on PEDOT:PSS-coated glass. PEDOT:PSS-coated glass
was used rather than PEDOT:PSS-coated MEA as the MEA devices consisted
of a solid gold electrode spin-coated with PEDOT:PSS and were opaque
and inaccessible to FRAP.

For all of the screening reactions, a fluorescent forked DNA substrate FORKF was prepared by annealing equal amounts of OLIGOA-BHQ2 (TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTACCCGATGTGTTCGTTC-BHQ2) and OLIGOB-TAMRA (TAMRA-GAACGAACACATCGGGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT) in equal amounts, boiling for 5 min and allowing the oligos to slowly cool to room temperature in the presence of 50 mM NaCl. For radioactive confirmation assays, ³²P-labeled FORKR was prepared by labeling 10 pmol of DC26 (5ʹ-TTTTTTTTTTTTTTTTTTTTTTCCCAGTAAAACGACGGGCAGTGC-3ʹ) with 30 μCi γ³²P-adenosine triphosphate (ATP) and T4 polynucleotide kinase (T4-PNK), passing through a G-25 spin column (GE Healthcare Life Sciences) and annealing to 25 pmol of TSTEM25 (5ʹ-GCACTGGCCGTCGTTTTACGGTCGTGACTGGGAAAACCCTGGCG-3ʹ) by boiling for 5 min followed by slow cooling to room temperature in the presence of 50 mM NaCl.