Part:BBa_K3001028

snR30 + RNA Mango II + T7 promoter

snR30-RNA Mango II is a snoRNA modified to contain the RNA aptamer RNA Mango II that is able to interact with the dye thiazole orange, and fluoresce very brightly. We were gifted this construct from the Kothe lab at the University of Lethbridge. The aptamer served as a target molecule for our in vitro experiments with the Cas13a enzyme isolated from Leptotrichia buccalis and the CRISPR RNA (crRNA) designed to interact with this part.

wetlab data

To characterize the parts for our diagnostic tool, we chose to work with the RNA aptamer, RNA Mango II. For further discussion on our choice of this part, please see our design page. Our team received an RNA Mango sample from the Kothe Lab at the University of Lethbridge. First, we PCR amplified the DNA (Figure 1). Then our team successfully completed in vitro transcription to convert the DNA sample into RNA (Figure 2). The Kothe Lab frequently works with run-off transcription so the RNA Mango sequence had no terminator, which increased the likelihood of other products in the sample. Therefore, our team chose to complete RNA purification using the method from the Panchapakesan et al., 2017 paper. (click here) This method uses streptavidin which binds very strongly to biotinylated thiazole orange. The resulting elutions have RNA that interacts with the biotinylated thiazole orange. Only RNA Mango should interact with the biotinylated thiazole orange, resulting in our sample being pure (Figure 3).

Figure 1. 1% agarose gel of PCR products of amplified RNA Mango. Left to right: lane 1: empty; lane 2: 1 kb ladder; lane 3: RNA Mango PCR product; lane 4: RNA Mango PCR product.

Figure 2. 8% Urea PAGE of in vitro transcribed RNA Mango. Left to right: lane 1:; lane 2:; lane 3:; lane 4:; lane 5:; lane 6: in vitro transcribed RNA Mango; lane 7: RNA Mango after DNase; lane 8: size standard from Kothe lab.

Figure 3. 8% Urea PAGE of streptavidin and biotin purified RNA Mango. Left to right: lane 1: in vitro transcribed RNA Mango; lane 2: wash 1; lane 3: wash 3; lane 4: elution 1; lane 5: elution 3; lane 6: elution 5; lane 7: elution 8; lane 8: elution 11; lane 9: elution 12; lane 10: elution 13.

After receiving a question regarding contamination and false positives, our team conducted an RNA Mango RNase Assay. Common RNases in the environment introduce the risk of false positives so we incubated RNase A or saliva with varying concentrations of RNA Mango. Regardless of the concentrations of RNase used, all RNA Mango was cleaved (Figure 4). Therefore, our system has a high likelihood of contamination. In an attempt to combat this issue, our team conducted an RNA Mango inhibitor assay.

Figure 4. 10% Urea PAGEs of RNA Mango and varying concentrations of RNase A incubated for increasing lengths of time at 37 ℃. (A) Left to right: lane 1: 2.5 μg/mL RNase A, 90 min incubation; lane 2: 2.5 μg/mL RNase A, 60 min incubation; lane 3: 2.5 μg/mL RNase A, 30 min incubation; lane 4: 2.5 μg/mL RNase A, 0 min incubation; lane 5: 1 μg/mL RNase A, 90 min incubation; lane 6: 1 μg/mL RNase A, 60 min incubation; lane 7: 1 μg/mL RNase A, 30 min incubation; lane 8: 1 μg/mL RNase A, min incubation; lane 9: 0 μg/mL RNase A, 90 min incubation; lane 10: 0 μg/mL RNase A, 60 min incubation; lane 11: 0 μg/mL, 30 min incubation; lane 12: 0 μg/mL RNase A, 0 min incubation; lane 13: water control; lane 14: negative control; lane 15: positive control. (B) Left to right: lanes 1-3: empty; lane 4: 10 μg/mL RNase A, 90 min incubation; lane 5: 10 μg/mL RNase A, 60 min incubation; lane 6: 10 μg/mL RNase A, 30 min incubation; lane 7: 10 μg/mL RNase A, 0 min incubation; lane 8: 7.5 μg/mL RNase A, 90 min incubation; lane 9: 7.5 μg/mL RNase A, 60 min incubation; lane 10: 7.5 μg/mL RNase A, 30 min incubation; lane 11: 7.5 μg/mL RNase A, 0 min incubation; lane 12: 5 μg/mL RNase A, 90 min incubation; lane 13: 5 μg/mL RNase A, 60 min incubation; lane 14: 5 μg/mL RNase A, 30 min incubation; lane 15: 5 μg/mL RNase A, 0 min incubation.

Our team wanted to see if adding an inhibitor would decrease the likelihood of contamination. To test this we incubated RNA Mango with the RNase inhibitor Murine. We determined that this did not affect the cleaving of RNA Mango. Our team recognizes that this may impact the results of the detection system. One way to combat this would be purifying the RNA from the sample prior to using the detection system. Commercial kits are available to do this from <a href=”https://www.thermofisher.com/order/catalog/product/AM1901#/AM1901”> ThermoFisher</a> and <a href=”https://bitesizebio.com/43279/isolating-bacterial-rna-from-blood/”> Bitesize Bio </a>. In the future, we hope to look into developing a simple RNA purification method that would work with our system. Additionally, we received feedback from <a href=”https://2019.igem.org/Team:Lethbridge_HS/Public_Engagement”>aGEM</a> to incorporate unnatural nucleotides at the ends of the RNA Mango sequence. This may prevent or limit any environmental RNases from cleaving the RNA Mango.

Figure 5. 10% Urea PAGEs of RNA Mango samples with Murine RNase Inhibitor (NEB) and RNase A after incubating for different time points at 37 °C. (A) Left to right: lane 1: positive Control; lane 2: negative control; lane 3: water control; lane 4: 0 U Murine, no RNase A; lane 5: 0 U Murine, 0 min incubation; lane 6: 0 U Murine, 30 min incubation; lane 7: 0 U Murine, 60 min incubation; lane 8: 0 U Murine, 90 min incubation; lane 9: 0.5 U Murine, no RNase A; lane 10: 0.5 U Murine, 0 min incubation; lane 11: 0.5 U Murine, 30 min incubation; lane 12: 0.5 U Murine, 90 min incubation; lane 13: 0.5 U Murine, 60 min incubation; lanes 14-15: empty. (B) Left to right: lane 1: 1 U Murine, no RNase A; lane 2: 1 U Murine, 0 min incubation; lane 3: 1 U Murine, 30 min incubation; lane 4: 1 U Murine, 60 min incubation; lane 5: 1 U Murine, 90 min incubation; lane 6: 2.5 U Murine, no RNase A; lane 7: 2.5 U Murine, 0 min incubation; lane 8: 2.5 U Murine, 30 min incubation; lane 9: 2.5 U Murine. 60 min incubation; lane 10: 2.5 U Murine 90 min incubation; lane 11: 5 U Murine, no RNase A; lane 12: 5 U Murine, 0 min incubation; lane 13: 5 U Murine, 30 min incubation; lane 14: 5 U Murine, 60 min incubation; lane 15: 5 U Murine, 90 min incubation.

Our team conducted a Cas13a activity assay to test the effectiveness of our enzyme. We incubated 300 nM of Lbu Cas13a complexed with the crRNA with various concentrations of RNA Mango to see the change in fluorescence of the dye thiazole orange. By analyzing our results, we can see that the fluorescence for the RNA Mango at the concentration 25nM sample decreased. This likely indicates that the Cas13a enzyme is cleaving. However, for higher concentrations, there was not a significant change in fluorescence. This may be due to having an insufficient amount of enzyme for proper cleaving to occur or our enzyme not being active enough to cleave larger amounts of RNA Mango in the same amount of time as the 25 nM sample. The sample with concentration 100 nM is excluded from these generalizations. We believe there may have been an error made in the reading. Another reason for the differences seen in the concentrations of RNA Mango could be caused by if our purified RNA is taking on multiple conformations that affect its ability to interact with thiazole orange. If the G Quadruplex is not forming properly, we would not see fluorescence. Graph C shows the results of our control experiment. Similar to the previously mentioned experiment we incubated 300 nM of Lbu Cas13a complexed with the crRNA with various concentrations of RNA, however this time we used RybA. This was a negative control experiment to test the specificity of our CRISPR Cas13a system. It seems that there was no significant change in fluorescence over time thereby indicating that no RybA was cleaved and our CRISPR Cas13a is specific. Since there was no added fluorescence molecule, it is also hard to interpret what any changes would be. Figure 6. Determining activity of Lbu Cas13a by targeting snR30 containing RNA Mango II by detecting a loss of fluorescence. Excitation occurred at 510 nm and emission at 535 nm and scans were completed for 3 hours. Data was normalized by dividing by the negative control, which contained all components except for RNA. (A) Raw scans normalized to the negative control of snR30-RNA Mango II at various concentrations in complex with Lbu Cas13a and the crRNA (n=1). Controls are also shown of only snR30-RNA Mango II, the target molecule and Lbu Cas13a, and the target molecule and RNase A (n=2 +/- SD for controls). (B) Relative fluorescence at the maximum fluorescence of snR30-RNA Mango II at 72 minutes (n=1 for snR30-RNA Mango II at various concentrations, n=2 +/- SD for controls). (C) Controls for activity of Lbu Cas13a and crRNA. RybA was used as a specificity control for cleavage (n=2 +/- SD for all data shown).

To confirm the effectiveness of our Cas13a enzyme we ran before and after fluorescence scanning samples on a urea page. Additionally, this would allow us to better interpret our experimental controls. This confirmed our speculation that the 25 nM sample was indeed cleaved by Lbu Cas13a. For the other samples, no significant difference is seen between the before and after samples. For the 50 nM before sample, we believe there may have been a loading issue that affected the quantification results seen in table 1. Our controls seen in Figure 7 B and C show that there are no significant changes between before and after scanning the samples. This means that are assay is specific for our targeted molecule needing to be present to have enzyme activation. Cas13a will also not cleave our target molecule without the crRNA in the complex.

Figure 7. 10% Urea PAGEs of Lbu Cas13a activity assay components. (A) Left to right: lanes 1-3: empty; lane 4: RNA Mango 100 nM post-scan; lane 5: RNA Mango 100 nM pre-scan; lane 6: RNA Mango 75 nM post-scan; lane 7: RNA Mango 75 nM pre-scan; lane 8: RNA Mango 50 nM post-scan; lane 9: RNA Mango 50 nM pre-scan; lane 10: RNA Mango 25 nM post-scan; lane 11: RNA Mango 25 nM pre-scan; lane 12: RNA Mango + RNase A post-scan; lane 13: RNA Mango + RNase A pre-scan; lane 14: negative control (no target or control RNA) post-scan; lane 15: negative control (no target or control RNA) pre-scan. (B) Left to right: lane 1: RNase A + RybA pre-scan; lane 2: RNase A + RybA post-scan; lane 3: RybA 25 nM pre-scan; lane 4: RybA 25 nM post-scan; lane 5: RybA 50 nM pre-scan; lane 6: RybA 50 nM post-scan; lane 7: RybA 75 nM pre-scan; lane 8: RybA 75 nM post-scan; lane 9: RybA 100 nM pre-scan; lane 10: RybA 100 nM post-scan. (C) Left to right: lane 1: crRNA pre-scan; lane 2: crRNA post-scan; lane 3: RNA Mango pre-scan; lane 4: RNA Mango post-scan; lane 5: RybA pre-scan; lane 6: RybA post-scan; lane 7: Cas13a Lbu pre-scan; lane 8: Cas13a Lbu post-scan; lane 9: Cas13a Lbu + crRNA pre-scan; lane 10: Cas13a Lbu + crRNA post-scan; lane 11: Cas13a Lbu + RNA Mango pre-scan; lane 12: Cas13a Lbu + RNA Mango post-scan; lane 13: Cas13a Lbu + RybA pre-scan; lane 14: Cas13a Lbu + RybA post-scan. Lane 15: empty.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 621
  Illegal SpeI site found at 161
  Illegal PstI site found at 1080
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 556
  Illegal SpeI site found at 161
  Illegal PstI site found at 1080
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 573
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 621
  Illegal SpeI site found at 161
  Illegal PstI site found at 1080
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 621
  Illegal SpeI site found at 161
  Illegal PstI site found at 1080
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1051