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Wednesday, May 10, 2006


There are trends in science. RNAi (short for RNA interference) is one of the latest trends. The story is that DNA makes RNA which makes proteins which make us. Since we have a hard time following a protein around and seeing what it's doing, we try to stop the protein from doing anything. In the past we have created "knock-out" mice, which lack the gene that codes for a protein. We have made antibodies go proteins so that they'll bind to and block the protein. We have thus been able to look at the world without the protein. What happens when a protein is taken out of the picture? Does the organism need it? Is it compensated for by the upregulation of another protein? RNAi is the latest tool in asking these types of questions. It is also much faster than making antidodies or knock-out mice.

There once was a Biotechnology company called Ribozyme Pharmaceuticals Inc. They failed. RNAi came along and they reinvented the company, "at the forefront of the effort to create RNAi-based therapies." They describe the technology as follows:

RNAi is a natural, selective process for turning off genes. RNAi is triggered by short interfering RNA (siRNA). An intermediate in the RNAi process in which the long double-stranded RNA has been cut up into short (~21 nucleotides) double-stranded RNA. The siRNA stimulates the cellular machinery to cut up other single-stranded RNA having the same sequence as the siRNA. molecules that engage a group of cellular proteins called RISC RISCRNA-Induced silencing complex, a protein siRNA complex that can recognize and destroy target mRNAs. (RNA induced silencing complex). The RISC guides the siRNA to its target messenger that is copied from a gene with the intention of being translated by ribosomes into a protein molecule. (mRNA mRNAmessenger RNA– the messenger between DNA DNADeoxyribonucleic acid. The primary genetic material of the cell, consisting of two long chains of nucleotides twisted together into a double helix. The sequence of the nucleotides (A, G, C, and T) in the DNA defines the genetic code for the organism; the sequence is copied and maintained through the complementary pairing of nucleotides (A with T, and G with C) across the strands of the double helix.and proteins) to destroy the mRNA. The process is extremely specific and enables siRNA to break up the mRNA associated with a disease-causing gene geneThe basic unit of genetic information, which is coded in the characteristic string of nucleotide bases (coded as A, C, T and G), in a specific sequence that provides the information usually for making a specific protein.or virus.

Okay so siRNA is a short piece of RNA that attaches itself to a RISC complex and directs the nucleic acid destroying enzyme to mRNA. The mRNA is chopped up and the protein doesn't get made. There are many questions regarding the actual mechanism of action. Does siRNA have to be double or single stranded? Is there any logic behind what fragment of the mRNA must be homologous with the siRNA? Does one piece of siRNA lead to many other pieces (after the mRNA is chopped up) which leads to more mRNA destruction? How does it end?

It is known that the effect is quite often short lived. The siRNA must be delivered into a cell which has lead to every researcher in the field testing various transfection reagents. The papers on delivery have yet to establish a coherent explanation of how this will work as a drug. Some targets are knocked down to a greater degree than others. The effect lasts longer in some targets than others. The RISC complex is made up of proteins. What happens if you try to knock out one of those proteins?

The entire field began back in 1989 when researchers were trying to make a purple petunia more purple by adding RNA that codes for purple. What they got was a white petunia. The color was eliminated and thus, they concluded that they knocked out the gene. Anti-sense had also been shown to knock out plant colors. The mechanism there is different. The entire anti-sense strand of an mRNA is attached to the mRNA molecule thus blocking it from being translated. (why wouldn't siRNA do this?) Later siRNA was shown to knock out genes in mammalian cells and the biotech world was off to the races. Money came in and careers and companies (like Ribozyme Pharmaceuticals) were revived. Papers on how RNAi works have chimed in on what the rules are for developing a good siRNA. The papers go into details of molecular modifications that make siRNA molecules more effective. Companies like
Qiagen, Invitrogen, Dharmacon and Sigma have all cashed in on the Biotech appetite for expensive little pieces of RNA and the assays needed to do the research.

Years later, there is no drug on the market. There is no definitive experiement that explains the mechanism. siRNA is going the way of gene therapry and anti-sense technology. Once again we've been fooled! Was it the marketing departments of Qiagen, Invitrogen, Dharmacon and Sigma? Were companies like Ribozyme Pharmaceuticals desparate for a new approach to RNA therapy? It seems as if we do not really know enough about the basics of nucliec acids inside a cell to really describe what is happening. We know that protein production is regulated. How, for example, is a protein downregulated? How does the cell know if it has enough protein? By the mRNA levels? Could this be effected by short pieces of the mRNA? What have we learned about the measurements of proteins and mRNA? Can we use RT PCR to acurately measure mRNA levels? The answer is no. Why not? Shouldn't RT PCR be corralated to protein levels? If not, what does that tell us about our understanding of the quantitation of nucleic acids and protein production?

There are so many questions left open for the scientific mind. Something is happening but what. Biotech companies are very simple in their approach to research. Whether they've chosen antibody technology, siRNA or some other small molecule approach, all they do is select a target and throw the technology at it. The details are left up to junior personel who don't stand a chance. It's too simple. The real science can only be done by those who don't care about the effects, but rather the measurements. Are they real and can they fit some mathematical model? The research could at least answer questions on the outside, such as how can we measure protein production? Tartrate resistant alkaline phosphatase for example is upregulated as osteoclast precursors fuse into an osteoclast. Prior to witnessing with your own eyes the final formation of the osteoclasts, the TRAP production stops. Can we find a way of predicting exactly when the production goes down. Do the enzyme activity measurements corrolate with enzyme production? How is the activity stopped? How is production shut down? So many questions and all basic and all fascinating if you think that the world inside of a cell is the great unknown.

Inside a cell there are little pockets of highly acidic solutions. There are scaffolds where RNA conducts the production of proteins. There are shuttles that take the proteins where they need to go. There is DNA twisting around making more DNA and RNA. There is a time when the whole production needs to focus on making another cell just like itself. To simply call up Invitrogen and order some siRNA against a target and have some kid mix it all up and run an assay and give you a PowerPoint slide show with some Excel charts does not constistute science. So far siRNA has given us jack squat and I predict that we will never see any real results from this latest trend. We may see a drug on the market but that doesn't count. Until we start studying our methods of measuring what happens inside a cell, we can't know what siRNA, or anti-sense is really doing. Even antibodies against proteins are a mystery. A recent paper on anti-TNF alpha showed that the antibody lead to an increase in protein production. Was the measurement accurate? Did the antibody cause more protein to be made because the protein interacting with the antibody was tied up? So many questions. They may be naive questions but those are usually the best ones. The big brained scientists sometimes get too fancy. The big funded scientists have gotten way to simple. The questions we attempt to answer should be simple. The answers may or may not be simple. That's the fun of trying to find things out. You never know how it will play out. What goes on in a cell? We just don't know yet.

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