TITLE: Folding of FluoroCubes
AUTHORS: Stefan Niekamp, Nico Stuurman, Ronald D. Vale

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Photobleaching limits extended imaging of fluorescent biological samples. Here, we developed DNA based “FluoroCubes” that are similar in size to the green fluorescent protein (GFP), have single-point attachment to proteins, have a ~60-fold higher photobleaching lifetime and emit ~50-fold more photons than single organic dyes. We demonstrate that DNA FluoroCubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometer precision.</div></div>

[STEPS]
?. [Design]
Pick a dye that you would like to use for the six dye FluoroCube. We have tested ATTO 488, ATTO 565, ATTO 647N, Cy3, Cy3N (sulfonated Cy3), and Cy5. Based on our experience, ATTO 647N and Cy3N work very well as six dye FluoroCubes and we recommend using these two dyes.
?. [Design]
Order the modified oligonucleotides from your favorite oligonucleotide synthesis company. Note, that different companies may use different linkers to attach the dyes to the oligos. This in turn may result in a different performance of the FluoroCubes because the linker length (and attachment chemistry) likely plays a role in achieving high photostability. We list all modified oligonucleotides used in this study and their respective manufacturer in Supplementary Table 1. To pick the right sequences for your desired FluoroCube, look at Supplementary Table 2. In Supplementary Table 2 we list which modified oligonucleotides from Supplementary Table 1 you need to purchase in order to create the desired FluoroCube.
?. [Folding ]
Dissolve each oligo in deionized water to a final concentration of 100 μM. Make 5 μl aliquots for each oligo and store at -20° C.
?. [Folding ]
For a 50 μl folding reaction: Take 5 μl of each of the four oligos required to fold the desired six dye FluoroCube (10 μM final) and add it to a PCR tube. Then add 25 μl of deionized water, and 5 μl of the 10x folding buffer (50 mM Tris pH 8.5, 10 mM EDTA and 400 mM MgCl2).
?. [Folding ]
Run your samples in a thermocycler to fold the six dye FluoroCubes with the following temperature ramp (it takes about 15 hours total):Denaturation at 85° C for 5 min Cooling from 80° C to 65° C with a decrease of 1° C per 5 minFurther cooling from 65° C to 25° C with a decrease of 1° C per 20 min Hold at 4° C.
?. [Purification ]
Folding products are analyzed by 2.0% agarose gel electrophoresis in TBE (45 mM Tris-borate and 1 mM EDTA) with 12 mM MgCl2 at 70 V for 3 hours. The gel box should be placed in an ice bath to minimize potential unfolding. After the gel finished running, it can be analyzed by any multicolor gel imager (e.g. Typhoon scanner). Note, do not add any DNA-stain such as ethidium bromide as it will bind to the DNA and therewith might interfere with absorbance and emission pattern of the FluoroCubes in downstream imaging applications.
?. [Purification ]
The desired band can then be extracted from the gel using a razor blade. It is typically the top band (See Figure 1c in the  BioRxiv manuscript). Afterwards the excised band is placed in a Freeze ’N Squeeze column (BioRad: 732-6165) and centrifuged for 3 min at 12,000 rcf at 4° C. The flow-through contains the desired FluoroCube. The FluoroCubes can be stored at 4° C for a couple of weeks.
?. [Introduction]
This protocol shows how the six dye FluoroCubes (described on BioRxiv) are designed, folded and purified. This protocol can not only be applied to the six dye FluoroCubes but also to all other DNA nanostructures described in the manuscript such as the Single Dye Cubes (FluoroCube with one dye), the double-stranded DNA constructs with one dye as well as the Compact Cubes with one or six dyes. The only differences between these designs will be the oligonucleotides used to assemble each construct (as described in detail in Step 3 and the attached Supplementary Tables).For simplicity, we will only use the six dye Fluorocube as an example throughout the protocol.