Team:Michigan/Design

Design

While there are many genetic switches for detecting proteins, for example, the lac operon and tet regulators, to our knowledge, every genetic switch is unique to the protein it detects. Aptapaper seeks to create a switch that could have the same logic adapted to any protein, similar to how toehold switches can be adapted and respond to any sequence of trigger RNA. Toehold switches refer to an RNA switch that works by sequestering the ribosomal binding site and start codon in a hairpin, while leaving a portion of what the RNA trigger binds to exposed in a “toehold” region. This toehold region dramatically changes the kinematics of the system, making it much easier for the trigger to bind. After the initial binding, the hairpin is “unzipped” by the trigger¹. Exact design specs of the well optimized second generation toehold design are detailed in Figure 1. Because toehold switches can respond to any RNA trigger, Aptapaper attempted to use aptamers in conjunction with toehold switches to allow efficient and sensitive protein sensing. See different design approaches below.

Figure 1: Forward Engineered Switches Inspiring Aptapaper Switch Design A.) RNA switch without trigger forms a stable hairpin structure, featuring a 15nt toehold region at 5’ end, 18nt total hairpin stem, 3nt AUG bubble (yellow), 15nt bubble sequestering 8nt ribosome binding site (green). Bubbles for AUG and ribosomal site allow switch to be activated by RNA triggers with any sequence. B.) RNA trigger (red) easily binds exposed toehold region. C.) RNA trigger binds to hairpin region of switch, forcing hairpin to unbind. D.) RNA trigger has fully bound switch, exposing the ribosome binding site so translation can be initiated1.

Switch 1.0 - Trigger-Aptamer Release for Toehold

Figure 2: Switch 1.0 Design A.) Protein of specificity is not present, so aptamer (blue) binds trigger (red). Toehold switch in hairpin conformation sequesters ribosome binding site. B.) Protein of specificity (gray) binds aptamer (blue), forcing trigger (red) to be released. Trigger can then easily bind exposed toehold region of switch. C.) Trigger binds to hairpin region of switch, forcing hairpin to unbind. D.) Trigger has fully bound switch, exposing the ribosome binding site so translation can be initiated.

In this design, a DNA trigger is bound by a DNA aptamer and “junk” DNA. The aptamer binds the protein of specificity when it is present, displacing the trigger, which can then activate the toehold switches previously discussed. While the system was based on the same DNA release sequence as validated for protein detection via fluorophore unquenching (FRET)², the 5’ 3’ orientation is reversed so that no part of toehold region is unbound when hybridized to the trigger. The toehold region of the switch used in our system is also 12nt instead of the 15nt seen in forward engineered switches for the same reason. Additionally, this design used a DNA trigger to activate the RNA switch, a previously untested configuration.

Because the toehold binding region of the trigger is complementary to the aptamer, this design necessitates that an RNA sequence identical to a portion of the DNA aptamer is exposed in the toehold region of the switch. It was unclear if the RNA version of the DNA aptamer would maintain some affinity for thrombin. The results (link) from this design suggest that the toehold region may in fact be functioning as an aptamer, which immediately suggests the two designs seen below.