Loading buffer

Various dehydration time was tested for dQD660 preparation. In a typical experiment, thiolated ssDNA (51 nt) (sequence: 5′-thiol-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA TTG TGA CAG CTG GAT CGT TAC-3′) was added to octadecylamine capped QD660 at a molar ratio of 500:1 in the presence of 100 mM NaOH to reach a final volume of 50 μl. Such solution was sonicated around 5 min and then was immediately combined with 600 μl of 1-butanol followed by a quick vortex for several seconds. After 0, 30, 60, or 90 min of incubation and shaking, 200 μl of 0.5×TBE buffer was added to the above solution followed by another quick vortex and a brief centrifugation at 2000g for several seconds to facilitate a liquid phase separation. DNA-functionalized QD660 were then recovered as a sublayer of the resulting two immiscible liquids. DNA-functionalized QD660 sample without purification (15 μl) was combined with 3 μl of 6× loading buffer (New England Biolabs) and loaded to a 1% agarose gel with 0.5× TBE. Each gel was run at 65 V for 60 min in 0.5× TBE at 4°C. Gels were then visualized under blue light transilluminator.

To investigate the impact of dehydration volume ratio on DNA density per QD, different 1-butanol/water volume ratio was used to prepare the dQD660 during dehydration process. In a typical experiment, thiolated ssDNA (51 nt) (sequence: 5′-thiol-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA TTG TGA CAG CTG GAT CGT TAC-3′) was added to octadecylamine capped QD660 at a molar ratio of 500:1 in the presence of 100 mM NaOH to reach a final volume of 50 μl. Such solution was sonicated around 5 min and then was immediately combined with 50, 300, 450, 600, or 750 μl of 1-butanol followed by a quick vortex for several seconds. Subsequently, 20, 100, 150, 200, or 250 μl of 0.5×TBE buffer was added to the above solution followed by another quick vortex and a brief centrifugation at 2000g for several seconds to facilitate a liquid phase separation. DNA-functionalized QD660 were then recovered as a sublayer of the resulting two immiscible liquids. DNA-functionalized QD660 sample without purification (15 μl) was combined with 3 μl of 6× loading buffer (New England Biolabs) and loaded to a 1% agarose gel with 0.5× TBE. Each gel was run at 65 V for 60 min in 0.5× TBE at 4°C. Gels were then visualized under blue light transilluminator.

The Fur-box binding activity of E. coli Fur was also analyzed using the hinfI site protection assay (18 (link)). Briefly, the Fur-box in the E. coli iucABCD promoter (5′-GAGAATCATTAGCATTCGC-3′) which contains the restriction hinfI site (5′-GAATC-3′) was synthesized (GenScript co) and inserted into plasmid pUC19 via BamHI and HindIII sites to create pUC19-iuc. Binding of Fur to the Fur-box protects the hinfI site from being cleaved by HinfI (18 (link)). For the hinfI site protection assays, pUC19-iuc (3.2 nM) was preincubated with Fur proteins (0–2.0 μM) in 10 μl reaction solutions containing MgCl₂ (2 mM), NaCl (150 mM), bovine serum albumin (0.1 mg/ml), and Tris (20 mM, pH 8.0) for 10 min at room temperature. Restriction enzyme HinfI (1.0 unit) (New England Biolab co) was then added to the reaction solutions. After incubation at 37 ^°C for 10 min, the reaction was stopped by adding 2 μl 6× loading buffer (New England Biolab co). The digested DNA products were separated by 1.5% agarose electrophoresis gel containing ethidium bromide (0.1 μg/ml) in 0.5X TAE (Tris-acetate-EDTA) buffer, run at 120 V for 35 min. The gel images were taken using the Kodak Gel Logic 200 Imaging System. The intensities of the DNA bands on the agarose gel images were quantified using ImageJ (NIH).

The Fur-box binding activity of E. coli Fur was analyzed using the hinfI site protection assays (9 (link)). The Fur-box in the E. coli iucABCD promoter (5′-GAGAATCATTAGCATTCGC-3′) contains the restriction enzyme HinfI site (5′-GAATC-3′). Binding of Fur to the Fur-box protects the hinfI site (highlighted) from being cleaved by HinfI (9 (link)). The iucABCD promoter was synthesized (GenScript co.) and cloned into plasmid pUC19 via BamHI and HindIII sites to create pUC19-iuc. For the hinfI site protection assays, pUC19-iuc was preincubated with purified Fur proteins in 10 μl reaction solutions containing MgCl₂ (2 mM), NaCl (150 mM), bovine serum albumin (0.1 mg/ml), and Tris (20 mM, pH 8.0) for 10 min at room temperature. Restriction enzyme HinfI (0.5 unit) (New England Biolab co.) was then added to the reaction solutions. After incubation at 37 ^°C for 10 min, the reaction was stopped by adding 2 μl 6× loading buffer (New England Biolab co). The digested DNA products were separated on 1.5% agarose electrophoresis gel containing ethidium bromide (0.1 μg/ml) in 0.5× TAE (Tris-acetate-EDTA) buffer, run at 120 V for 35 min. The gel images were taken using the Kodak Gel Logic 200 Imaging System. The intensities of the DNA band on the agarose gels were quantified using ImageJ (NIH).

SDD-AGE analysis was performed as previously described (79 (link)). Briefly, harvested cells resuspended in cytosolic extraction buffer (Abcam) were incubated on ice for 10 min and subjected to dounce homogenization. The homogenate was clarified at 1000g for 10 min at 4 °C. The supernatant was further centrifuged at 10,000g for 30 min at 4 °C to pellet the intact crude mitochondria. Crude mitochondria were resuspended in 6× loading buffer (New England Biolabs) and loaded into a vertical 1% agarose gel (Lonza). Electrophoresis was performed in running buffer (1× TAE and 0.1% SDS) for 40 min with a constant voltage of 100 V at 4 °C. The proteins were transferred to an Immobilon membrane (Millipore) and subjected to IB analysis.

For Tet-QD600, Tet wireframe DNA origami objects with QD wrapping domain were incubated with QD600 (molar ratio was 1:4, in 1× TAE with 20 mM MgCl₂) and undergo a secondary annealing protocol (55 °C for 10 min, 55 °C down to 45 °C for 2 min/°C, 45 °C down to 25 °C for 10 min/°C). To assemble Pep-QD660, pentagonal pyramid wireframe DNA origami objects with a QD wrapping domain were incubated with QD660 (molar ratio was 1:4) at room temperature overnight. The mixture (15 μL) was then combined with 3 μL of 6× loading buffer (NEB) and loaded to a 0.8 % agarose gel with 1× TAE and 12 mM MgCl₂ and 1× SYBR Safe (ThermoFisher, Waltham, MA). Each gel was run at 65 V for 40 min in 1× TAE with 12 mM MgCl₂ at 4 °C. Gels were then visualized under blue light and UV light for Tet-QD600, and under blue light for Pep-QD660.