Part:BBa_K4170026
crRNA targeting the miR-17-3P (extra loop 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-17-3P, 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-17-3P (standard design-BBa_K4170023) but has a different design for the spacer sequence. This extra hairpin loop has been reported to reduce the off-target effects and undesired interactions of crRNA sequence with the target RNA sequence. (Ke et al., 2021)The effect is described in Figure 2. The difference in Gibbs free energy between the off-target (undesired) interaction and the on-target interaction is increasing with the extra hairpin loop in the 3’ end of the spacer sequence. Bigger the difference between 2 energy states the more favorable will be the interaction with the minimum free energy and in this case the on-target interaction. (Ke et al., 2021) The number of the nucleotides introduced to the 3’ end of the spacer sequence is 5, aiming for the formation of 5 base pairs and the number of nucleotides on the stem designed to be 4. This means that the 5 introduced nucleotides are reverse complementary to the nucleotides to position 43-47.
Sequence- crRNA 17-3p-hairpin 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 17-3p. 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). This specific crRNA sequence have a different design for the spacer sequence. The design of crRNA sequence based on the introduction of an extra hairpin loop in the 3’ end of spacer sequence.
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 -10.4 kcal/mol suggesting a relative stable structure. 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 0.46 is low since values >0.5 are preferable in terms of stability. 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 -4.4 kcal/mol is the result of hydrogen bonds in the spacer sequence that designed to formed in 3’end, but since the value is low the miRNA-crRNA sequence interaction is energetically favorable. 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 -40.3 kcal/mol of the binding energy is nearly 4 times more than the MEF of the crRNA sequence then the stability of complex is the preferable energetical state. 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 1.0 is showing that the binding on the spacer sequence will be exactly as the suggested binding upon designing the sequence. The extra introduced nucleotides did not affect the position of the interaction. 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
17-3p-hairpin 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 -380.66 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 57.7 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 Domain and the interaction site of the crRNA sequence is both the Repeat and the Detect Part of the crRNA 17-3p-hairpin. 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. The final step of the in-silico analysis is the molecular dynamics (MD) simulations. The MD simulations investigate the stability of the system and can measure the interaction energy between the protein and the crRNA sequence. Utilizing the same pdb files from the docking process, the MD simulations proceeded with the GROMACS software(Lindahl, Hess and van der Spoel, 2001). Using the pdb file of LbuCas13a and crRNA sequence and then after using the Amber force field, we utilized the pdb2gmx function of the GROMACS for the generation of topology for 2 the molecules. After the steps of the ionization and solvation, we equilibrated the system with respect to temperature and pressure. After these steps, the simulation began. The simulations of the systems were for a total time of 1 ns with time step of 1 ps. The first aspect of the data analysis is the Root Mean Square Deviation for the crRNA sequence. The Root Mean Square Deviation is representing the difference between two structures: a target structure and a reference. For the MD simulations for checking the system’s stability, we evaluate the difference between the stable final structure and the initial structure. The low values of RMSD shows the stability of the complex after its formation.
The other analysis that took place was the calculation of the Root Mean Square Fluctuation. The Root Mean Square Fluctuation is a calculation of individual residue flexibility, or how much a particular residue moves (fluctuates) during a simulation. It can be observed that some nucleotides in spacer part demonstrates higher values in comparison with the Repeat part meaning that the main interactions between protein and crRNA take place with the Repeat Part. Overall, the values for both sequence’s parts are low meaning that all nucleotides are involved in the protein/RNA interaction.
The next step of the analysis is the calculation of the Hydrogen Bonds formed with the crRNA sequence. The Hydrogen bonds are some of the most stable bonds that can be formed between 2 molecules. This is depicting their importance in biological molecules. The number of the hydrogen bonds seems to be stayed the same after 450 ps meaning that the complex reaches its stability before the end of the simulation.
Finally, in the next figure is demonstrated the binding energy of the LbuCas13a and the crRNA over time. The mean value of the binding energy calculated to be -1447.59 kJ/mol with standard deviation +- 160.99 kJ/mol. The main energy that contributed was the Coulomb interactions meaning that the nature of the interactions between the two molecules are mainly electrostatic.
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.Ke, Y. et al. (2021) ‘Hairpin-Spacer crRNA-Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP) Identification’, Advanced Science, 8(6), pp. 1–11. Available at: https://doi.org/10.1002/advs.202003611.
2.Lindahl, E., Hess, B. and van der Spoel, D. (2001) ‘GROMACS 3.0: A package for molecular simulation and trajectory analysis’, Journal of Molecular Modeling, 7(8), pp. 306–317. Available at: https://doi.org/10.1007/S008940100045.
3.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.
4.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.
5.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.
6.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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