Difference between revisions of "Part:BBa K4286115"

Revision as of 17:26, 11 September 2022

shRNA molecules that can silence the gene encoding RNA pol III subunit C6.

We found that the coding sequence of RNA pol III is a housekeeping gene that has been applied to inhibit Rhizoctonia solani. We searched the cDNA database of Rhizoctonia solani AG1-IA according to the sequence provided by the literature, and found the homologous cDNA sequence of AG1-IA strain.

Next，the RNA pol III subunit C6（RhiXN_03964） sequences found by this method were analyzed, queried or predicted on the National Center for Biotechnology Information (NCBI) website whether there were multiple spliced versions of mRNA, and if there were, the homologous region was taken as the target region of RNAi interference target. However, it may be due to the lack of relevant research and literature support, and the corresponding mRNA has no variant. Also，the total nucleic acid database blast was carried out on the target CDS to query the similarity of homologous genes in adjacent species, and shRNA was designed in non-conserved regions to improve the species specificity of our shRNA and ensure biological safety.

And then by siRNA Design websites (http://rnaidesigner.thermofisher.com/rnaiexpress/setOption.do?designOption=shrna&pid=8294778592286769918) to analyze these gene fragments. The program scans the DNA sequence of a gene fragment and calculates the binding energy of sense and antisense siRNAs based on the sequence pattern. The higher the score, the stronger the binding ability of the siRNA to the target sequence. Based on the principle of shRNA design, we selected the fragments with high potential siRNA activity from a series of sequences. At the same time, we try to select shRNA fragments that inhibit the target in the first third of the length of the target mRNA. Because according to the mechanism of RNAi, the dsRNA synthesised is longer, producing more small siRNAs subsequently. It can better amplify the secondary inhibition effect.

For biosafety， the candidate RNAi fragments were submitted to the total mRNA database for blast, and the sequence similarity was compared. Focus on species with more than 90% similarity and their nucleic acid fragments to ensure that there is no matching of common species (human, rice, dog, wheat, etc.) to ensure the specificity of the sequence.

Finally, the chosen RNAi fragment is assembled in the order of RNAi fragment — loop — the reverse complement of the sense RNAi fragment.

Usage and Biology

Assembly

Characterization

Sequencing

Sequence and Features