Difference between revisions of "Part:BBa K5335002"
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<p>The 19 bp stem-loop structure can be specifically bound by MS2 CP. We designed a tandem stem-loop structure capable of serving as a transport adaptor for future loading of other RNA.</p>
 
<p>The 19 bp stem-loop structure can be specifically bound by MS2 CP. We designed a tandem stem-loop structure capable of serving as a transport adaptor for future loading of other RNA.</p>
 
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<center><b>Figure 1. Secondary structure simulation results of tandem stem-loop structures, simulated using SnapGene.</b> </center>
 
<center><b>Figure 1. Secondary structure simulation results of tandem stem-loop structures, simulated using SnapGene.</b> </center>
 
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<p>The stem loop structure can bind specifically to MS2 coat protein and is encapsulated in VLP. We plan to use this as an anchor sequence for the inner envelope RNA in the future, so we preliminarily verified its binding activity by this assay. By reviewing the literature, we refer to a method for the preliminary detection of protein binding to nucleic acid. It has been documented in the literature that MS2 VLP can be completely eluted during the Ni-NTA column purification process <b><sup>[1]</sup></b>. We mixed the purified sample 1:1 with TNE buffer (V/V) and then mixed it with DNA loading buffer containing glycerol for electrophoresis in 1% agarose gel. After electrophoresis, the gel was stained with 1:10,000 dilute safe red dye solution, and the results were observed and recorded. The gel was then stained with G250 dye for protein components, and the strip location was recorded after decolorization with decolorization solution. The results showed that the chromogenic site of nucleic acid was the same as that of protein, which could preliminatively indicate the existence of RNA and capsid protein, so that RNA was not hydrolyzed by enzyme (<b>Figure 1.</b>).</p>
 
<p>The stem loop structure can bind specifically to MS2 coat protein and is encapsulated in VLP. We plan to use this as an anchor sequence for the inner envelope RNA in the future, so we preliminarily verified its binding activity by this assay. By reviewing the literature, we refer to a method for the preliminary detection of protein binding to nucleic acid. It has been documented in the literature that MS2 VLP can be completely eluted during the Ni-NTA column purification process <b><sup>[1]</sup></b>. We mixed the purified sample 1:1 with TNE buffer (V/V) and then mixed it with DNA loading buffer containing glycerol for electrophoresis in 1% agarose gel. After electrophoresis, the gel was stained with 1:10,000 dilute safe red dye solution, and the results were observed and recorded. The gel was then stained with G250 dye for protein components, and the strip location was recorded after decolorization with decolorization solution. The results showed that the chromogenic site of nucleic acid was the same as that of protein, which could preliminatively indicate the existence of RNA and capsid protein, so that RNA was not hydrolyzed by enzyme (<b>Figure 1.</b>).</p>
 
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<img src=https://static.igem.wiki/teams/5335/ms2-spy-e/nuc-acid-and-pro-stain.png width=100% alt="">
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<img src=https://static.igem.wiki/teams/5335/ms2-spy-e/new-agarose-pro-and-rna.png width=100% alt="">
 
<center><b>Figure 2. Gel retardation analysis different staining conditions.</b> The left image is the result after dyeing with Safe Red dye, and the right image is the result after dyeing with G250 dye. Line 1: DNA loading buffer. Line 2: DNA loading buffer + TNE buffer. Line 3: Purified protein + TNE buffer + DNA/RNAase + DNA loading buffer. Line 4: DNA loading buffer + TNE buffer + DNA/RNAase</center>
 
<center><b>Figure 2. Gel retardation analysis different staining conditions.</b> The left image is the result after dyeing with Safe Red dye, and the right image is the result after dyeing with G250 dye. Line 1: DNA loading buffer. Line 2: DNA loading buffer + TNE buffer. Line 3: Purified protein + TNE buffer + DNA/RNAase + DNA loading buffer. Line 4: DNA loading buffer + TNE buffer + DNA/RNAase</center>
 
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Revision as of 10:44, 30 September 2024


It can bind to MS2 capsid protein and encase in VLP

It can bind to MS2 capsid protein and encase in VLP. It is used to verify the packaging effect of the binding sequence.


Usage and Biology

The 19 bp stem-loop structure can be specifically bound by MS2 CP. We designed a tandem stem-loop structure capable of serving as a transport adaptor for future loading of other RNA.

Figure 1. Secondary structure simulation results of tandem stem-loop structures, simulated using SnapGene.

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]

Gel retardation analysis

The stem loop structure can bind specifically to MS2 coat protein and is encapsulated in VLP. We plan to use this as an anchor sequence for the inner envelope RNA in the future, so we preliminarily verified its binding activity by this assay. By reviewing the literature, we refer to a method for the preliminary detection of protein binding to nucleic acid. It has been documented in the literature that MS2 VLP can be completely eluted during the Ni-NTA column purification process [1]. We mixed the purified sample 1:1 with TNE buffer (V/V) and then mixed it with DNA loading buffer containing glycerol for electrophoresis in 1% agarose gel. After electrophoresis, the gel was stained with 1:10,000 dilute safe red dye solution, and the results were observed and recorded. The gel was then stained with G250 dye for protein components, and the strip location was recorded after decolorization with decolorization solution. The results showed that the chromogenic site of nucleic acid was the same as that of protein, which could preliminatively indicate the existence of RNA and capsid protein, so that RNA was not hydrolyzed by enzyme (Figure 1.).

Figure 2. Gel retardation analysis different staining conditions. The left image is the result after dyeing with Safe Red dye, and the right image is the result after dyeing with G250 dye. Line 1: DNA loading buffer. Line 2: DNA loading buffer + TNE buffer. Line 3: Purified protein + TNE buffer + DNA/RNAase + DNA loading buffer. Line 4: DNA loading buffer + TNE buffer + DNA/RNAase

[1]Williams LA, Neophytou A, Garmann RF, Chakrabarti D, Manoharan VN. Effect of coat-protein concentration on the self-assembly of bacteriophage MS2 capsids around RNA. Nanoscale. 2024 Feb 8;16(6):3121-3132. doi: 10.1039/d3nr03292b.