LOCKING MECHANISM 1:

TARGET RECOGNITION

OVERVIEW

To control the conformation of H1 and H2 single hinges and by extension, EHSs, a locking mechanism was incorporated into the design of the hinges that could lock the hinges in an open or closed conformation. 

This experiment aims to confirm whether the components of the locking mechanisms are able to recognize their target sequence. Target recognition was assessed through polyacrylamide gel  electrophoresis (PAGE) analysis.

BACKGROUND INFORMATION

Figure 1. The mechanism for TMSD, adapted from [1].

Toe-hold mediated strand displacement (TMSD) is a process to exchange a single strand of DNA from a DNA double helix for a new single strand of DNA. As shown in Figure 1 [1], the complementary region between two strands forms a DNA double helix; however, one strand is multiple base pairs longer than the complementary strand creating an overhang, which is called the toe-hold region. An input strand is introduced that is complementary to the toe-hold region and has a sequence with greater binding affinity to the downstream strand in the complementary region. Due to this greater binding affinity, the input strand migrates along the complementary region, displacing the existing strand. The output is a new DNA double helix with a single strand of DNA as waste. TMSD forms the basis of our locking mechanism design.

Figure 2. Hinge locking mechanism.

Starting with the closed conformation, a section of the padlock is bound to the lock strand. A key strand can be introduced, which recognizes the toe-hold region of the padlock, and displaces the lock strand. Without the lock strand, the padlock strand is fully stretched allowing the hinge to be in the open conformation. An anti-lock strand can be added to the system to remove lock strands by binding to the lock strand. Additionally, the lock cannot displace the key as it does not have a higher binding affinity to the padlock than the key. The only method to remove the key and allow the hinge to change to the closed conformation is to add an anti-key strand into the system. The anti-key has the strongest affinity to the key strand, therefore, the anti-key strand first binds to the toe-hold region of the key and then displaces it from the padlock strand. The key and anti-key pair can then be removed. With free padlock strands in the system, the lock strand can be reintroduced, or used from previously bound ones in the system if the anti-lock was not added, to bind the padlock causing the closed conformation. The cycle is now reset and can be repeated continuously. 

 H1 and H2 hinges each contain binding sites for two padlock strands located in the middle of the bricks of each hinge. The locations were chosen to allow for the locking mechanism to be applied to the hinges without blocking the binding sites for EHS assembly. The four padlock strands are identical, except for their two ends used to bind to the hinge, which are not involved in the locking mechanism sequence. 

 In our experiment, we refer to hybrids when a strand binds to its complementary strand in the locking mechanism sequence. Possible hybrids include:

1. Lock + padlock
2. Padlock + key
3. Key + anti-key
4. Lock + anti-lock 

METHODS

Validation of locking mechanisms target recognition.

Strands of the locking mechanism were first assessed for whether they were able to recognize and bind to their intended target in the locking mechanism sequence. This was assessed by creating samples, shown in Table 1, that contained different combinations of strands in the mechanism sequence in a 1:1 molar ratio. An example preparation for 10 μL samples is shown in Table 2.

Table 1: Sample Labeling Code. “x” indicates which component was added to the sample. *One of H1 padlock 1 or 2 or one of H2 padlock 1 or 2 was added. 

Table 2: Sample preparation for target recognition validation. 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. 

Samples were then incubated at 30 °C for 25 min. To serve as a reference for relative band migration, 1 μL of  GeneRuler Ultra Low Range DNA Ladder (ThermoFisher) at a concentration of 100 ng/μL. A minimum of 20 ng of each locking mechanism strand was run in a separate lane and served as the negative controls. The negative controls were made based on Table 3 and mixed with 2 μL of 6X loading dye.

 Table  3: Preparation of negative controls.

All four padlock strands are shown in Table 3. Only one padlock control was included per set of controls which corresponded to the padlock being assessed for its ability to recognize its target strands in the locking mechanism sequence.

To create a 20% polyacrylamide gel, the reagents in Table 4 were combined and allowed to solidify at room temperature. After samples and controls were loaded, the gel was run at 105 V for 2 hours and then stained in 1X Sybr Gold solution for 10 min before being imaged on a GelDoc.

Table 4: 20% PAGE gel preparation.

RESULTS

Figure 3: A 20% polyacrylamide gel testing whether strands and their targets in the locking mechanism are bound together using samples with H1 Padlock 2 as the padlock from Table 2 and controls from Table 3. 

The banding patterns of the sample lanes imply that the strands of the locking mechanism are able to recognize and bind to their targets in the mechanism sequence as intended. The distance that each unbound strand of the locking mechanism migrates down the gel is shown through the negative controls. The location of these bands were used to determine which bands were representative target binding in the sample lanes.

L: Only one band was observed at the location of the padlock strands between 50 and 75 bp. This indicated that all of the lock strands have bound to a padlock strand. Further validation of lock and padlock hybridization can be found in Supplementary Information: Experiment 3.

LL: Two bands were present. The upper band represented the padlock strands. The lower band between 25 and 35 bp was expected to be the lock strands and anti-lock strands hybridized together. The lock and anti-lock hybrid indicated that the presence of a padlock strand does not interfere with lock and anti-lock binding. 

LK: Two bands were present. The presence of a band above the padlock strands between 75 and 100 bp indicated that the key strands were able to hybridize to the padlock strands. This band also indicated that the presence of a lock strand does not interfere with key and padlock hybridization. Furthermore, a lack of band at the location of the unbound key strands suggested that all key strands hybridized. A fainter band present at the location of the padlock strands was still observed.

LKK: When the anti-key strands were added a third band between 35 to 50 bp appeared representing the hybridization of the key and anti-key. Key and padlock hybrids were still seen. 

LKLK: In this lane, lock and anti-lock, as well as key and anti-key, hybrids were both seen. The presence of multiple locking strands simultaneously in solution did not prevent the lock and key from recognizing their anti-strand counter parts. 

K: Key and padlock hybrids were observed between the 70 bp and 100 bp ladder mark.  

The same banding pattern was observed for the remaining three padlock strands. Lastly, it was observed that despite the same ng of DNA loaded into each control lane, locking mechanism strands with higher molecular weight resolved more clearly. 

DISCUSSION

As expected, each strand of the locking mechanism was able to recognize their complementary target strand. In lanes that contained key and padlock, both key and padlock hybrids, as well as unbound padlocks, were observed. The co-occurrence of bands containing these hybrids and unbound strands suggested that that key and padlock hybridization was not 100% efficient. For future experiments, more key strands could be added to exceed a 1:1 ratio and favor greater hybridization of key and padlock strands.

 In sample LKK, both the key and anti-key were added at the same time and both contained complementary regions to the key. Therefore, it was expected that both padlock and key hybrids, as well as key and anti-key hybrids, would form. More key and anti-key hybrids were expected due to a greater binding affinity to each other, which was observed based on visual inspection of band densities.  

In lanes expected to contain unbound lock strand, we suspect they are present; however, they may be close to or below the lower limit of DNA required to be loaded into the gel for detection. This may be because samples were prepared to maintain a 1:1 molar ratio with the padlock strands compared to controls that ensure a minimum 20 ng of strand per lane. Our expectation that unbound lock strand was still present in these samples was confirmed by the presence of lock and anti-lock hybrids in other sample lanes. The same concentration of lock strands was added to each sample made to contain them. The appearance of lock and anti-lock hybrids would only occur if both strands were present, therefore, we conclude that lock strands in unbound form or otherwise were still present.