Part:BBa_K4170028
crRNA targeting the miR-17-5P (mismatch design)
This part contains the sequences of the stem loop (repeat part) and the spacer (detection part) required for the formation of the CRISRPR/Cas13a complex and the hybridization with the mature miR-17-5P, respectively. The LbuCas13a protein adopts a bilobed structure which consists of a a-helical REC lobe and a NUC lobe. The crRNA stem loop sequence is anchored to the space between the NTD and Helical-1 domain forming extensive contacts between the crRNA and the LbuCas13a protein. This crRNA derives from the crRNA targeting the miR-17-5P (standard design-BBa_K4170027) after the introduction of an artificial mismatch at the position 16.The nucleotide that replaced the corresponding one at the spacer sequence is its complementary. Based on the literature the artificial mismatch leads to a decrease at the fluorescence signal from the trans cleavage activity of the protein, when the crRNA interacts with a different RNA sequence compared to the target RNA.[2]
Sequence- crRNA 17-5p-mismatch design and in silico evaluation
The sequence is consisted of 2 main parts. The first part is called Repeat Part and it is a standard nucleotide sequence that binds to the Recognition Lobe of LbuCas13a and forms a stable complex with it. The second part is called spacer or detection part and it is responsible for the detection of the miR 17-5p. The Repeat part contains 31 nucleotides, and the spacer sequence is designed to contains 20 nucleotides that are complementary to the target miR sequence(Liu et al., 2017). After the selection of the 20 nucleotides of the spacer sequence, we introduced an artificial mismatch at the position 16.
The first step of the in-silico evaluation is the evaluation of its thermodynamic properties. The software used for this task is ViennaRNA(Lorenz et al., 2011. The properties studied were : Minimum Free Energy, GC content of the sequence, Linearity of Detection Part, the Binding energy with the target miRNA and perfect matches.
The Minimum Free Energy is a measure of stability of the RNA and the negative it is the more stable will be. The Vienna Software is calculating the energy based on the secondary optimal structure. The value of -10.2 kcal/mol and a probability of the Minimum Free Energy in the ensemble is 0.64 or 64% suggesting a stable secondary structure that is adopted from the majority of the crRNA molecules. GC content is ratio of the Guanine and Cytokine that are present in the sequence. The higher the ratio, the more stable the sequence will be. This phenomenon is based on the different chemical groups that are present in every nucleotide resulting stronger hydrogen bonds in comparison with other nucleotides and the value 0.48 is low in terms of stability since a ratio > 0.5 is preferable. Linearity Of Detection Part (LODP) shows the secondary structure of the detection part of the crRNA sequence. Closer to 0 more linear will this part be. The linear structure of the detection part of crRNA is highly associated with the proper function of the LbuCas13a protein. The value of -4.1 kcal/mol is the result of unwanted hydrogen bonds in the spacer sequence and based on this energy value may lead to system’s dysfunctionality. Binding energy is the energy term of the difference between the Gibbs free energy of the complex RNA and the free energy of crRNA and miRNA target. The binding energy is calculated using the secondary structure of the sequences. Since the value -36.7 kcal/mol of the binding energy is nearly 4 times the MEF of the crRNA sequence then the stability of complex is the preferable energetical state and the binding is predicted to be immediate. Perfect Matches is a measure of evaluation of the RNA complex that is formed by crRNA and miRNA target sequences. The secondary structure of the complex is calculated through RNAduplex, and perfect matches is the ratio of the nucleotides that are bind properly in detection part of the crRNA sequences. The ratio of 0.95 (19/20) is showing that the binding on the spacer sequence will be exactly as the suggested binding upon designing the sequence. The spacer sequence designed to pair with the miRNA with 19 bonds since it has an artificial mismatch. The secondary structure of the miRNA after binding with the sequence that predicted from RNAduplex is:)))).))))))))))))))) that ) shows a pair nucleotide and . shows an unpaired one. The predicted secondary structure of the crRNA sequence and the complex formed between the crRNA and miRNA sequence are demonstrated on the next figure:
The next step for the in-silico evaluation is the molecular docking with the LbuCas13a protein. For this process, the utilized pdf file for LbuCas13a acquired from Protein Data Bank (ID 5XWY) and the pdf file for the crRNA 17-5p-mismatch sequence generated through the RNAComposer Sever. The docking algorithm was the HDOCK docking algorithm for the webserver(Zhang Di Yumeng Yan, 2017). The docking model selected was the best model that had the more negative value based on the scoring algorithm of HDOCK server(Wang et al., 2020).
The docking score is the energy score calculated through the scoring function of HDOCK algorithm.[4] The docking score is negatively related to the stability of the complex, meaning the more negative is the docking score more stable the complex receptor-ligand will be. The value of -349.98 kcal/mol shows that the complex formation is the energetical favorable state. The Ligand Root Mean Square Deviation is showing the difference between the positions of nucleotides of the ligand’s structure before and after docking. The ligand RMSD value of 49.91 Angstrom shows a major change in the conformation of the ligand which is acceptable since the ligand in this specific docking process is an RNA sequence of 51 nucleotides. The interaction site of the LbuCas13a based on the best model from HDOCK docking algorithm is the NUC Lobe and the interaction site of the crRNA sequence is the Repeat part of the crRNA 17-3p-mismatch. In comparison with the literature (Liu et al., 2017), the protein-RNA interactions are on the REC Lobe of the protein and the Repeat Part of the crRNA sequence suggesting that the docking model resulted to a different conformation of the 2 molecules.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Citations
1.Liu, L. et al. (2017) ‘The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a’, Cell, 170(4), pp. 714-726.e10. Available at: https://doi.org/10.1016/j.cell.2017.06.050.
2.Lorenz, R. et al. (2011) ‘ViennaRNA Package 2.0’, Algorithms for Molecular Biology, 6(1), pp. 1–14. Available at: https://doi.org/10.1186/1748-7188-6-26.
3.Shan, Y. et al. (2019) ‘High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity’, Analytical Chemistry, 91(8), pp. 5278–5285. Available at: https://doi.org/10.1021/acs.analchem.9b00073.
4.Wang, L. et al. (2020) ‘Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein’, Theranostics, 11(2), pp. 649–664. Available at: https://doi.org/10.7150/thno.51479. Zhang Di Yumeng Yan (2017) ‘HDOCK : a web server for protein-protein and protein -DNA/RNA docking based on a hybrid strategy’.
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