Difference between revisions of "Part:BBa K4586005"

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Revision as of 21:02, 11 October 2023

MCP-ADAR2

Part Description

This part codes for the catalytic portion of ADAR2 (adenosine deaminases acting on double-stranded RNA) that converts adenosines (A) to inosines (I) in both coding and non-coding RNA transcripts. Through a process known as hybridization or deamination, In addition to conjugating this part to MCP (MS2 coat protein), which has an affinity for the MS2 hairpin structure.

Usage

This part is implemented in our design to increase the effectivity and sensitivity of our therapeutic agent, as changing the status of the DART V ADAR switch from off to on is mediated through the ADAR enzyme by deamination of the mismatched adenosine group into an inosine group. Thus, the stop Codon UAG within the sensor of the switch will be converted into UIG, and the translation of the therapeutic agent will precede expressing Cas12k and MCP-ADAR2. Thus, MCP-ADAR2 is creating a positive feedback loop that amplifies the signal transmitted from different cargo copies present within the target cell as shown in figure 1.

Literature Characterization

The study tested the action of sensors containing MS2 hairpins without ADAR, with ADAR p150, or with MCP-ADAR2dd.

Characterization By Mutational Landscape

In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (Q173E) shows the highest epistatic fitness, while the lowest score was associated with the mutation (S51D).

Improvement by our team

MCP-ADAR2 is an improved version of mutant form of ADAR2 enzyme (BBa_K2818001) that was designed by (iGEM18_NTU-Singapore) We conjugated the mutant form ADAR2dd to MS2 coat protein MCP, which specifically binds to short MS2 RNA hairpin 3D structure that replaces the promiscuous dsRNA-interacting domain of natural ADAR. Integrating MCP-ADAR and flanking the sensor UAG codon with two MS2 hairpins allows us to amplify the initial signal of our therapeutic cargo and replace the low background editing of natural ADAR by the targeted and efficient MCP-ADAR2 which ensures maximum activity of our therapeutic cargo within the target auto-reactive B-cells as shown in the following figure.

Experimental Characterization

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P10) including MCP-ADAR2, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.

We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P10).

References

Gayet, R. V., Ilia, K., Razavi, S., Tippens, N. D., Lalwani, M. A., Zhang, K., ... & Collins, J. J. (2023). Autocatalytic base editing for RNA-responsive translational control. Nature Communications, 14(1), 1339. Sequence and Features