HINGE EXTENSION

OVERVIEW

The goal of this experiment was to assess whether H1 and H2 units could polymerize together and form large structures we call Extended Hinge Systems (EHS)s. To accomplish this we purified H1 and H2 single hinge units and incubated them in the presence of neighbor strands and polymerization strands. The presence of EHSs after incubation was assessed using agarose gel electrophoresis. Multiple bands present in EHS lanes at fixed intervals indicated the assembly of EHS structures.

BACKGROUND INFORMATION

Polymerization stands are single-stranded DNA species used to bind H1 and H2 units together. These polymerization strands range from 28 to 62 bp long and are the out-most strands that bind to the end of the hinge bricks. Part of the polymerization strand is complementary to the scaffold located at the end of an H1 hinge brick. The other part of the polymerization strand is complementary to the H2 scaffold. This allows H1 and H2 to bind brick to brick in an alternating series. Neighbor strands, coined by Dr. Lauback, are a type of staple strand that are located next to the polymerization strands. 

Under the guidance of Dr. Stephanie Lauback, Associate Professor at Hillsdale College, neighbor strands and polymerization strands were left out of single hinge assembly. This step was taken to prevent single hinge units from clumping together during assembly, as bundles of blunt end double helices at the end of structures can cause their aggregation. Leaving these strands out of single hinge assembly results in single-stranded loops in the scaffold and the end of the hinge arms that prevent aggregation. After single hinge assembly and purification these strands can be added back to the structures for EHS assembly. We define EHSs as any structure that consists of more than one hinge bound together.

Figure 1. CADnano view of neighbor staples and polymerization strands from [1].

Amicon spin column purification was used for the purification of H1 and H2. Amicon spin column purification is the technique of utilizing centrifugal force and filters to separate DNA products by size and shape. The Amicon centrifugal filters have ultra-small pores that do not allow for anything bigger than 100 kDa through. Therefore, when purifying single hinges or EHSs, only staple strands theoretically are the ones that can pass through, while assembled hinges and EHSs are collected. Centrifuges allow the separation of particles based on the size and shape as the centrifugal force applied when the rotor spins pulls particles toward the outer ring of the rotor, forcing them to pass through the filter. A washing step with a buffer is added to increase the removal of smaller DNA species and other possible contaminants. The purified DNA is collected by inverting the filter in a new column and centrifuging again, where this time the centrifugal force pulls the sample to the bottom of the column, collecting it. The purified DNA can then be used for downstream experiments.

Issues with Amicon spin column purification include excess staple strands being caught with larger structures and clogging in the filter, preventing more staple strands from getting removed and continuing to allow staple strands to be collected from assembled hinges and EHS in the final product. An additional issue is that with more times the hinges get washed with a buffer, they can get pulled deeper into the filter, get stuck, and not get pulled out in the inversion step. And with excessive washes, the filter could get worn out and tear, resulting in greater losses. Therefore, to minimize these issues, optimization of the number of washes is needed.

METHODS

Purification 

To assemble EHSs, H1 and H2 single hinge units were purified using spin column purification to separate the assembled hinges from excess staple strands left over from single hinge assembly. 

This protocol was adapted from the work of Lauback et al. [1].

1. 100 kDa Amicon Ultra Centrifugation filters (MilliporeSigma) were equilibrated by centrifuging 500 μL of resuspension buffer (0.5X TAE, 4 mM MgCl2) at 5000 rcf for 15 min. Flow through was discarded. 
2. Assembled hinge samples were topped up to 500 μL with suspension buffer and then added to the column and spun at 2000 rcf for 30 min. Flow through was discarded. 
3. A wash step was then performed by adding 500 μL of resuspension buffer and centrifuging the column at 2000 rcf for 30 min. Additional wash steps could be added to limit the amount of remaining excess staples.
4. To collect the purified hinges, the column was inverted into a new tube and centrifuged at 1000 rcf for 2 min. Purification yielded 20 – 25 μL of purified samples. 
5. Purified samples were nanodropped and the ratio of scaffold to staple molecular weight was used to infer the concentration of purified hinges.

To confirm whether excess staple strands were removed, purified samples were run on a 1% agarose gel as previously described in Single Hinge Assembly. Negative controls included the p8064 scaffold as well as unpurified H1 and H2 samples.

EHS Assembly

To assemble EHSs, the following components were mixed together and incubated at 45 °C and cooled by 2 °C every hour until reaching 4°C. The volume of purified H1 and H2 added was dependent on the concentration of single hinges recovered during extraction. As purification consistently yielded 20 – 25 μL, the concentration of purified hinges varied with the volume of assembled hinges initially added to the purification column. H1, H2, H1N, H2N, and polymerization strands were added in a 1:1:2:2:5 molar ratio, as shown in Table 1.

Table 1 Components for EHS assembly after H1 and H2 purification. 

To assess whether EHSs formed, samples were run on a 1% agarose gel as described in Single Hinge Assembly. EHSs were purified as described above for downstream experiments.

RESULTS

Figure 2. A) 1% agarose gel of before purification H1 and H2 single hinges B) Agarose gel of purified H1 and H2 single hinges.

Post purification, smearing below the 75 bp ladder mark was visibly absent in the lanes of purified H1 and H2 single hinge units, indicating the removal of excess staple strands. H1 and H2 single hinge units still resolved between 2000 and 3000 bp compared to 1500 and 2000 bp suggesting that purification did not impact the structural integrity of the hinges. The presence of residual staples as indicated by fainter smearing below the 75 bp mark was noted in some lanes. This issue was overcome by adding additional wash steps during purification.

Figure 3. 1% agarose gel showing various EHSs consisting of increase number of H1 and H2 single hinge units. 

Figure 4. 1 % agarose gel of with lane A containing EHS with padlock strands and lane B containing EHS without padlock strands.

All bands in EHS-lanes resolved at molecular weights (in reference to the ladder) higher than the p8064 negative control. In lanes that contained EHS samples (Figure 3), seven bands were observed and were characterized by increasing molecular weights with decreasing band densities. These bands were not present prior to incubating the components required for EHS assembly indicating that H1 and H2 hinges were successfully bound together. The lowest band in these lanes resolving between 2000 and 3000 bp is expected to be H1 and H2 single hinge units that have not reacted in the allotted incubation time. Each additional band above the band containing the unreacted single hinge units is representative of an EHS which can range from a series of two hinges (H1 and H2) bound together, to three hinges, and so forth.

DISCUSSION

Because H1 and H2 hinges were allowed to self-polymerize during the incubation ramp, EHSs containing different numbers of single hinges were possible. Although only seven bands could be resolved on the gel, it is suspected that even larger EHSs may be present, however in quantities less than the ng of DNA required for band resolution. The decreasing concentration of high order EHSs is expected as the probability of multiple binding events required to make larger EHSs is less than that required for a fewer number of hinges to bind together. We expect that if the incubation ramp is extended, this would provide a greater time frame for larger EHS species to form.

TEM imaging of EHS samples confirmed the formation of structures consisting of multiple hinges and matched the expected dimensions for those number of hinges bound together. These results will be published after the judging period in alignment with competition rules.