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Building Block of BUS
The Biosensing Unit Structure (BUS) is designed as revolute joint between two stiff arms and resembles a hinge-like structure, as shown in FIGURE 1. Each arm is designed as 3 x 6 square lattice. The two arms are joined together by 6 sections of 30nt DNA, connecting each helix of the extended middle layer of the arm. The outer layer is 87 nm while the shortest one is at the bottom measures 57nm. The BUS was designed with an extended outer layer in part to help distinguish orientation of the structure when viewed via TEM. The middle layer of the BUS is extended on the joint end to minimize steric hindrance and achieve a nearly 360° rotation of the arms when BUS is in open configuration.
Figure 1: Building Block of BUS. (A)The BUS consists of two arms connected at extended middle layer by 6 DNAs of length 30nt each (red lines). (B) Each arm is designed as 3 by 6 square lattice and different lengths of each layer shown in figure.

Each BUS incorporates 4 - 30nt latching strands (shown in blue, FIGURE 2). The latching strand contains an aptamer that recognizes specific protein. The aptamer can also bind to a complementary ssDNA that closes the BUS, as explained later.

The interior of each arm carries 10 - 8nt ssDNA overhangs. The different colors (yellow, black, green and orange) of the overhangs correspond to a different set of overhang sequences. Two different sequences were used for each internal face to ensure other structures would bind with the correct orientation to form a trimer. Thus, each BUS has total 20 overhangs or 4 different sets of overhangs as shown in FIGURE 2. The primary function of the internal overhangs is for trimer formation these can also facilitate closing of the BUS.

Figure2: Opened BUS. The interior of BUS showing 20 ssDNA internal overhangs of each 8nt in length (green, orange, yellow and purple). Different colors show different sequences of internal overhangs. Trimer formation is facilitated by these internal overhangs. The latching strands (shown in blue) are 30nt in length. The latching strands are designed to recognize and bind to specific protein. Also latching strand close the BUS when bind with closing ssDNA.
Closed BUS and Protein Recognition
The BUS can be closed by a 30nt palindromic ssDNA sequence that is complementary to 15nt on each latching strand on each BUS arm (FIGURE 3). The remaining 15 bases of a latching strand work as toehold for ssDNA recognition and binding. The extended outer layer of the BUS also serves to shield the latching strands and prevent binding of the closing strand while the structure is in an inverted configuration. ssDNA is used as proof-of-concept however the latching strands can be substituted with aptamer sequences for the detection of proteins or other nucleic acids.
Figure 3: Closed BUS. The BUS is closed when closing ssDNA binds to the latching strands of the BUS. The toe-hold region recognizes and binds to the protein. When protein is bound to toe-hold region, closing ssDNA is displaced by the protein through strand displacement mechanism.
The opening of the BUS is triggered by the binding of a target protein or oligonucleotide to the latching strands shown in FIGURE 4. In preliminary tests where the BUS is kept closed by a DNA strand bridging the two latching strands, an oligonucleotide complementary to the full length of the latching strands is used to displace the closing strand via toehold-mediated strand displacement. Disrupting the connection between latching strands returns the BUS to an open configuration and exposed the internal overhangs which enables the formation of trimers.
Figure 4: Actuation of BUS. (A)BUS is closed in the absence of opening strand. (B) Opening strands in solution which bind to latching strands.(C) The closing strands are replaced by opening strand and the BUS is Opened.
The BUS trimer is formed when three different opened BUSes are connected together as in the FIGURE 5. Trimer formation is dictated by the different sets of internal overhangs incorporated by the BUS as shown in FIGURE 2. To avoid forming secondary structure, each arm has two different sequence sets with 5 internal overhangs. If one arm of the BUS has an internal overhang sequence set A and the other arm had internal overhang sequence set B, then the BUS can be called BUS AB. Inclusion of a sequence set that is complementary to set A (denoted as A’) on a different BUS will allow these two structures to bind together. Thus, these 6 sequence sets can be used to form a trimer. Specifically, when open conformations of three BUSes (denoted as AB, B’C’, and CA’) are within vicinity of each in solution, they form the trimer AB:B’C’:CA’.
Figure 5: Three BUS AB, CA’ and B’C’ forming trimer through binding between internal overhangs.

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