16.10: RNAi Experimental

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the function of several genes without genetically modifying the organism.

Applications of RNAi

RNAi helps in analyzing gene functions. For example, the RNAi technique helped screen chromosomes I and III of C.elegans and led to the identification of genes involved in cell division and embryonic development. This technology has also been successfully applied to Drosophila melanogaster to identify genes with essential roles in embryonic development, biochemical signaling cascades, and other fundamental cellular processes. In coffee plants, the gene responsible for producing theobromine synthase was knocked out using an RNA construct, producing decaffeinated coffee plants. Research shows that small interfering RNAs (siRNA) can inhibit infections caused by human immunodeficiency virus, hepatitis B virus, and poliovirus in cultured human cell lines. Researchers have also successfully knocked down genes expressed by the respiratory syncytial virus responsible for causing severe respiratory disease in infants and neonates.

Advantages of RNAi over conventional gene knockout

Before discovering RNAi technology, gene function was analyzed by knocking out the gene of interest from the genome and observing the phenotypic changes. While gene knockout is an irreversible method, RNAi is a reversible method that provides large-scale silencing of protein-coding genes in a genome. Moreover, it is a precise technique that can differentially silence genes even with a single nucleotide variation. Therefore, it can help in targeting dominant mutants like some oncogenes. Moreover, the RNAi technique is highly potent as the effector molecules function at a low concentration compared to the oligonucleotides or ribozymes used in the old method.