LOCKING MECHANISM 2:

TARGET DISPLACEMENT

OVERVIEW

This experiment aims to confirm whether the locking mechanism’s components can displace their intended targets to open and close hinges in a cyclic manner. Target recognition was assessed through PAGE analysis.

BACKGROUND INFORMATION

This experiment builds off the work of Locking Mechanism: Target Recognition. Relevant background information for this experiment can be found by visiting that page. After validating the strands in the locking mechanisms can recognize their targets, we then evaluated if the targets could be displaced with an input strand complementary to the strand that the target was bound to.

METHODS

Lock strands and key strands were first incubated with padlock strands in a 1:1 molar at 30 °C for 25 min. This incubation step was taken to first anneal either a key or lock strand to a padlock strand. 

First incubation 

Sample 1: Lock  + padlock

Sample 2: Lock + padlock

Sample 3: Key + padlock

Table 1: Sample preparation. All volumes are in μL. The concentration of all locking mechanism strands were at 1000 nM and 100 mM MgCl2 was used in sample preparation. *One of H1 padlock 1 or 2 or one of H2 padlock 1 or 2 was added. 

Then, the next strand in the mechanism sequence that could displace the strand bound to a padlock strand was added and the samples were incubated again at 30 °C for 25 min. Adding these strands after the first incubation helped ensure that any targets that these strands bound to were first bound to the padlock strand. 1.82 μL of anti-lock, key, and anti-key, were added to the samples, which was the same volume of padlock added to the sample in the first incubation.

Second Incubation 

Sample 1: Lock  +  padlock + anti-lock

Sample 2: Lock + padlock + key 

Sample 3: Key + padlock + anti-key

Next, a 20% polyacrylamide gel was prepared as described in Locking Mechanism: Target Recognition. Negative controls were also prepared as described in Locking Mechanism: Target Recognition. 1 μL of a 100 bp ladder (100 ng/μL) was loaded as a reference. Samples were prepared for gel loading by mixing 4 μL of 6X loading dye with the 20 μL samples. The gel was run at 105 V for 2 hours, stained in 1X Sybr Gold solution for 10 min, and visualized on a GelDoc.

RESULTS

Figure 1: A 20% polyacrylamide gel testing whether the anti-strands are able to displace their target strands from the padlock strand using samples with H2 padlock 2.

As seen in Figure 1, when comparing the band patterns of lanes before and after the second incubation, the anti-lock, lock, and anti-key were able to displace their respective target from the padlock strands. The additional bands seen in samples after the second incubation matched the migration distance of the lock/anti-lock, key/padlock, and key/anti-key hybrids, as observed in Locking Mechanism: Target Recognition. The addition of the anti-lock to sample 1, produced lock and anti-lock hybrids, the addition of key to sample 2 produced padlock and key hybrids, and the addition of anti-key to sample 3 produced key and anti-key hybrids. Although the addition of the key in sample 2 was able to produce key and padlock hybrids, there was variability in the density of this band across the different padlock strands for H1 and H2 hinges (See Supplemental Information).

DISCUSSION

The variability in key and padlock hybrids across all of the H1 and H2 padlock strands suggests the key strand may not be able to efficiently displace the lock strand that has been bound to the padlock strand. However, the anti-lock strand was able to consistently produce lock and anti-lock hybrids. Hence the anti-lock strand could be added at this step to more effectively displace the lock from the padlock for the key to bind to the padlock instead. Future iterations of this experiment should add both anti-lock and key during the second incubation of sample 2.