amiRNA design |
Introduction Artificial microRNA (amiRNA) technology exploits microRNA (miRNA) biogenesis pathway to produce artificially designed small RNAs using miRNA gene backbone. It generates a single type of small RNA population all with the same selective nucleic acid sequence, usually 21 nucleotides (nt) in length, providing a feasible method for either silencing an individual gene or simultaneously silencing closely related gene isoforms. Artificial microRNA (miRNA)-directed gene silencing has advantages over traditional inverted-repeats gene silencing vector in more gene silencing specificity and less off-target effects. A dedicated web-based miRNA vecotor design interface was established to help the users to design their poly-cis miRNA-directed gene silencing constructs to silence multiple genes of interest. |
Steps Step 1: predict the target miRNAs from gene coding sequence. The input is the gene coding sequence and the output should be a set of artificial microRNAs which are of 21 nucleotides. In the very beginning, we need to select an appropriate backbone. Choosing a good backbone is heavily based on your requirement and your application. When we generate pattern library we should take the constraints which are from the template into account. sometimes we have to face such a problem: how to do if we get so many artificial microRNAs that we do not know which one or whose are good choices. Our solution is to let users run BLAST to go through the database and then to make a decision. Select the artificial microRNAs and run BLAST for the targeting specificity. Choose those artificial microRNAs that are only complementary to the target gene transcripts but have no complementarity to other gene transcripts genome-wide around the 5' end of those artificial microRNAs Step 2: predict artificial miR* sequence from miR. The first step for this task is to analyze the template and generate the translation table. it is difficult sometimes because it needs domain knowledge. Based on the translation table plus some specific rule, the program generates artificial miR* from miR. Step 3: predict primers from the secondary structure of pre-microRNA Firstly, the code reverse the artificial miRNA* to get P2. Then it get the complementary string T1 for artificial miRNA string. Lastly, reverse T1 to get P1. |