Mimetics and Inhibitors
The study of miRNAs and their function is greatly facilitated by the development of miRNA mimetics and inhibitors. To meet this experimental need, Dharmacon (www.dharmacon.com), part of Thermo Fisher Scientific (www.thermofisher.com), offers the Miridian® miRNA mimics and inhibitor collections. Miridian includes both inhibitors and mimetics of known miRNAs in human, mouse, and rat. They can be used to alter the balance of any particular miRNA and assess its effect on the cell. This was the topic of the presentation of Thermo Fisher’s Scott Baskerville, Ph.D. at the miRNA meeting.
“Unlike other miRNA mimic designs,” said Bill Marshall, Ph.D., vp for technology and business development, “these modifications inactivate the passenger strand of the Miridian mimic and ensure that it performs similarly to the endogenous mature microRNA without nonspecific passenger strand activity.”
After the double-stranded pre-miRNA is processed by the endonuclease dicer, the 20–25 nucleotide guide strand enters the RISC complex, where it pairs with its complementary mRNA and induces mRNA degradation, effectively turning off the gene. In the Miridian miRNA mimetics, a chemical modification insures that the passenger strand is inactivated and the correct strand enters the RISC complex.
In addition to developing a unique set of miRNA mimics, Dharmacon’ R&D team also performed detailed structure/function studies for development of the next generation of miRNA inhibitors. As detailed in a recent article in RNA, inhibitor potency and longevity are greatly affected by the annealing strength of miRNA inhibitors and the presence of structural elements outside the core (reverse complement) region of the inhibitor.
The company’s Miridian inhibitors adopt design strategies from both domains, using chemical modifications to strengthen inhibitor binding to the miRNA in the RISC complex and incorporating flanking structures around the modified complementary sequence to enhance overall functionality. These reagents were utilized in a recent phenotypic screen to successfully identify miRNA biomarkers that induce unique cellular states. Thermo Fisher is launching a miRNA profiling service based on the Miridian technology.
Tim Davison, Ph.D., senior scientist at Asuragen, discussed the company’s work in biofluid screening for miRNA biomarkers. It is not always clear in a tissue sample what is and is not tumor tissue, nor what the appropriately matched control samples should be. miRNAs are present in biofluids such as serum, plasma, saliva, and urine. These biofluids may potentially be used to define the signature or presence of cancerous tissue. To access biomarkers from fluids, the company developed and validated miRNA isolation methods and a microRNA expression analysis to face the unique sample preparation and analytical challenges presented by working with biofluids.
Asuragen is the successor to Ambion (www.ambion.com). In 2005, Ambion founder, Matt Winkler, sold the Ambion research reagent business to focus on miRNA-based diagnostics and therapeutics. The remaining Diagnostic and Services division was rolled into the new company, Asuragen.
Asuragen has used multiple expression analysis platforms, including qRT-PCR, mirVana™ bioarrays, as well as a custom Affymetrix (www.affymetrix.com) array that includes published miRNAs outside the Sanger miRBase content to screen its biofluids. “The intent was that if we look at the commonly recognized Sanger content and see a separation or depth of information in published microRNA content not found in typical microarrays, we will have the opportunity to screen a large number of microRNA for the potential identification of biomarkers,” said Davison, Ph.D.
“What we’ve been working on as well is understanding what the limitations are of the original sample source. Depending on what tissue you’re looking at, there are different amounts of microRNA you can actually isolate from these tissues; specifically in the context of optimizing isolation procedures and understanding the impact of normalization of array and qRT-PCR data.”
New tools and technologies have new frontiers in RNA research. The field of microRNA has exploded, with notable discoveries regarding their role in cancer and other disorders of development. However, some of the most important news is the rewriting of the rules of molecular biology and the breakdown of the central tenets. Scientists are entering uncharted waters, where the great unanswered questions of biology and human health could finally be adressed.
Asuragen’s Tim Davison speculated on some of the implications of miRNAs, including the possibility that SNPs could have a relationship with them. “This is a whole new realm of understanding,” he remarked. “Just when we thought we had it all figured out, along came microRNA.”