Difference between revisions of "Part:BBa K4586029"

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This composite part codes for our tissue-specific switch and consists of two elements: first, a stop codon (UAG) sensor that is designed to be complementary to a specific RNA within the target cell or tissue; in our case, our target is the auto-reactive B-cells that secrete ACPA, and the sensor is complementary to the variable chain of ACPA mRNA; second, a two-hairpin structure flanking the sensor, which is called MS2, that has binding affinity to MCP and is conjugated to the ADAR2 catalytic portion in order to enhance the sensitivity and amplify the signal of our therapeutic cargo (Cas12k).
 
This composite part codes for our tissue-specific switch and consists of two elements: first, a stop codon (UAG) sensor that is designed to be complementary to a specific RNA within the target cell or tissue; in our case, our target is the auto-reactive B-cells that secrete ACPA, and the sensor is complementary to the variable chain of ACPA mRNA; second, a two-hairpin structure flanking the sensor, which is called MS2, that has binding affinity to MCP and is conjugated to the ADAR2 catalytic portion in order to enhance the sensitivity and amplify the signal of our therapeutic cargo (Cas12k).
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<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts/ms2-sensor-mcp-adar-7.png
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>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.  </span></p></div></html>
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==Literature Characterization of sensor of the switch==
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The study investigated whether a DART VADAR sensor directed towards a point mutation of interest could specifically activate translation in cells expressing a disease biomarker. To this end, the study focused on the single-base mutation (c.658 T > C) in the human p53 tumor suppressor gene, which results in a Y220H substitution and is known to destabilize p53's DNA binding domain, making it a key factor in the development of breast cancer. The study co-transfected plasmids expressing either the wild-type or the Y220H mutant p53 gene with a DART VADAR sensor that is specifically made to detect the p53 mutant in HEK293FT cells.<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
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position: relative;
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top: 50%;
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transform: translate( -50%);
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"src="https://static.igem.wiki/teams/4586/wiki/parts/ntgivfnjtfkkuhorlip3q-transformed.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>The sensor was engineered to be entirely complementary to the Y220 codon and the surrounding sequence, preventing the target adenosine from being edited by ADAR. In cells expressing p53-Y220H, we noticed a fivefold activation of the reporter gene downstream of the sensor, demonstrating the selectivity of DART VADAR sensors.
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</span></p></div></html>
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==Literature Characterization of MS2==
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The study tested if MCP-ADAR activated the translation of cargo, specifically if the sensor contained MS2 RNA hairpins that encoded this cargo.
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<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
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width:75%;
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height:auto;
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position: relative;
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top: 50%;
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left: 40%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts/ms2-1.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>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. Orange points refer to the sensor with MS2, and blue points refer to the sensor without MS2. They found that MS2 increased the specificity of the switch.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 14:15, 12 October 2023


DART-V-ADAR (tissue specific switch)

Usage and Description

This composite part codes for our tissue-specific switch and consists of two elements: first, a stop codon (UAG) sensor that is designed to be complementary to a specific RNA within the target cell or tissue; in our case, our target is the auto-reactive B-cells that secrete ACPA, and the sensor is complementary to the variable chain of ACPA mRNA; second, a two-hairpin structure flanking the sensor, which is called MS2, that has binding affinity to MCP and is conjugated to the ADAR2 catalytic portion in order to enhance the sensitivity and amplify the signal of our therapeutic cargo (Cas12k).

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 of sensor of the switch

The study investigated whether a DART VADAR sensor directed towards a point mutation of interest could specifically activate translation in cells expressing a disease biomarker. To this end, the study focused on the single-base mutation (c.658 T > C) in the human p53 tumor suppressor gene, which results in a Y220H substitution and is known to destabilize p53's DNA binding domain, making it a key factor in the development of breast cancer. The study co-transfected plasmids expressing either the wild-type or the Y220H mutant p53 gene with a DART VADAR sensor that is specifically made to detect the p53 mutant in HEK293FT cells.

The sensor was engineered to be entirely complementary to the Y220 codon and the surrounding sequence, preventing the target adenosine from being edited by ADAR. In cells expressing p53-Y220H, we noticed a fivefold activation of the reporter gene downstream of the sensor, demonstrating the selectivity of DART VADAR sensors.

Literature Characterization of MS2

The study tested if MCP-ADAR activated the translation of cargo, specifically if the sensor contained MS2 RNA hairpins that encoded this cargo.

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. Orange points refer to the sensor with MS2, and blue points refer to the sensor without MS2. They found that MS2 increased the specificity of the switch.


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]