Hi Tuck,
Thanks for the responses and for looking into the targeting and delivery question. What is GFP (green florescent protein) anyway?
My siRNA filter dredged up the following "lay terms" explanation of how RNAi works, which I found a useful refresher, so thought I would post to the board.
Cheers,
Thomas
howestreet.com
RNA INTERFERENCE ~ Silencing Genetic Instructions
A cell—whether plant or animal—possesses DNA (deoxyribonucleic acid), which is in turn composed of two RNA (ribonucleic acid) molecules, or polypeptide chains ('peptide' is another term for protein). These chains, comprising our 46 chromosomes, constitute tens of thousands of genes, which in turn encode and control the development of many thousands of proteins.
But not all of these proteins are needed for every cell function. A cell must be selective in which genes it expresses or inhibits, and at what time. Also, our cells must protect themselves from viruses (small, 'rogue' strands of RNA) and other mobile genetic elements, which, if left unchecked, could take over its replication machinery and start producing viral proteins. Thus, over billions of years of evolution, cells developed molecular mechanisms for 'silencing' gene expression.
While evidence began mounting over a decade ago, it has only been in the past 3-4 years that one of these genetic "censor" mechanisms has been revealed and given serious study. Its called RNA interference (RNAi) and it works by intercepting and destroying only the messenger RNA (mRNA)—the 'copy me' instruction molecules—triggered by an invading virus, or mutated protein. RNAi is able to selectively destroy these pathogenic messages without interfering with other, necessary protein transcription.
RNAi has for several years been a potent research tool for geneticists working with plants and smaller animals like worms and fruit flies. These researchers have been able to utilize this natural cellular mechanism to suppress any gene they chose, thus allowing them to deduce a gene's specific function. Later research has revealed that double-stranded RNA—RNA segments that can fold back on themselves—seems to be the primary trigger for the RNAi effect. However, subsequent experiments with injecting these double-stranded RNAs into live animal cells resulted in triggering the cell's interferon response, which shut down all of the cell's genes. Researchers realized that a deeper understanding of RNAi was needed.
Further research revealed that selective gene silencing occurs when double-stranded RNAs are modified by two enzymes, dubbed 'Dicer' and 'Slicer'. The Dicer enzyme cuts the longer RNA molecule into smaller fragments known as 'short interfering RNAs' (siRNAs). These asymmetric, double-stranded pieces are then unwound and one strand is incorporated into a larger molecular aggregate called the RNA-induced silencing complex (RISC). The RISC uses each strand of siRNA to 'run interference', that is, the siRNA is positioned to bump into thousands of mRNAs that normally populate the cell nucleus. But the particular siRNA will only adhere to a mRNA whose nucleotide sequence (the string of amino acid bases labeled 'a', 't'. 'c', and 'g') closely resembles its own. Thus this 'silencing' mechanism is far more selective than the interferon mechanism. When a matching messenger RNA docks with the siRNA strand, the 'Slicer' enzyme comes into play, chopping it in two, thus rendering the mRNA incapable of protein transcription. The RISC remains intact and moves on to other silencing tasks.
This revolutionary knowledge of our cells' natural gene silencing mechanisms has sent a tidal wave of excitement through the bio-engineering and genetic sciences. RNA interference has of course attracted the attention of pharmaceutical companies that seek to develop a new class of medicines that interfere with the protein products of specific, cancer-causing genes (oncogenes). A half dozen labs have already had success in using RNAi to stop viral replication (HIV, polio, hepatitus C) in human cell cultures.
This past September, Acuity Pharmaceuticals, a company in Philadelphia founded in 2002, announced that the FDA had granted permission for it to conduct the first human test of RNA interference. These trials will use an RNAi inducing drug to target the gene that triggers the process of macular degeneration, an age-related deterioration of the retina that is the leading cause of blindness in the elderly.
Scientists believe they can 'co-opt' this mechanism to shut down any gene in the body. Some are experimenting with 'shuttle viruses' to deliver microRNA (another form of RNA that inhibits gene expression) into a cells' nucleus. But even with such a revolutionary discovery as RNAi, many years of additional trials and experiments will be needed before a safe and reliable gene therapy based upon RNAi is in widespread use. |
