Difference between revisions of "Part:BBa K4586005"

Latest revision as of 13:48, 12 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.

Figure 1: This figure illustrate the activity of our DART V ADAR tissue specific switch that is designed to be in the on state after recognition of the autoreactive B-cells,this recognition based on mismatched base editing in the level of transcribed RNA that is mediated through ADAR enzyme activity.

Literature Characterization

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

Off-state refers to mNeonGreen expression in the absence of iRFP720 trigger mRNA, while on-state refers to mNeonGreen expression in the presence of iRFP720 trigger mRNA. They found that constitutive expression of MCP-ADAR causes an increase in sensor activation in the absence of the trigger.

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).

Figure . An illustration of the effects of different mutations on the Epistatic Fitness of MCP ADAR.

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.

This figure illustrates the mechanism of MCP-ADAR recruitment toward the site of editing flanked by two MS2 RNA hairpin structures.

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).

After the ligation step, we cultured the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the pCDNA3(-) plasmid vector containing insert parts.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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