Extended Gene Silencing
Bio-Rad has collaborated with Integrated DNA Technologies to develop RNAi tool sets. The results of this two-year collaboration, which pairs IDT’s siRNA design and synthesis proficiency with Bio-Rad’s transfection and amplification expertise, delivers validated Dicer-Substrate 27-mer small interfering RNA (siRNA) duplexes for RNAi applications. “We’ll be presenting silencing longevity and potency data for Dicer-Substrate 27-mer siRNAs and matched 21-mer siRNAs,” reports Eli Hefner, Ph.D., senior scientist at Bio-Rad. “We will also be discussing our Dicer-Substrate 27-mer (siLentMer) product validation procedure,”
Dr. Hefner points out that not everyone has heard of synthetic Dicer-Substrates. Dicer-Substrate siRNAs (DsiRNAs), which have recently been employed for in vivo studies using intraperitoneal and intrathecal routes of administration, had their beginnings with John Rossi at City of Hope. In vivo, long dsRNAs are cleaved by the RNase III class endoribonuclease dicer into 21–23 base duplexes having 2-base 3´-overhangs. These species, called small interfering RNAs (siRNAs), enter the RNA-induced silencing complex and serve as a sequence-specific guide to target degradation of complementary mRNA species.
“Despite the continuous evolution of sequence selection algorithms, many synthetic siRNAs still fail to achieve the desired level of silencing,” adds Dr. Hefner. “The only way to truly know that your siRNA will work is to buy one that was pretested. Even when an siRNA is capable of reducing the target gene mRNA to low levels, the corresponding protein concentration might not be affected. This can occur when the window of effective siRNA operation is shorter than the protein half life, hence the desire to identify RNAi-inducing molecules such as the siLentMer Dicer-Substrates that have a longer window of operation.”
Bio-Rad’s siLentMer-validated siRNAs help solve this dilemma by delivering effective gene silencing for an extended period of time using low siRNA concentrations, the company reports. According to Dr. Hefner and senior product manager Christina Whitman, they are functionally tested via RT-qPCR for 85% mRNA knockdown, and many will demonstrate sustained silencing for up to 6–9 days.
“We validate by testing in-house, and we are stringent with our passing criteria,” says Whitman. “For some experiments, synthetic siRNAs are able to achieve 70 percent silencing, which might be okay depending on your protein stability. With the validated siLentMer Dicer-substrates you are guaranteed the highest possible silencing of 85 percent or higher.”
siRNA Specificity Enhancement
In the last two years, Applied Biosystems has focused on improving siRNA by improving specificity, says Susan Magdaleno, Ph.D., senior manager, scientist at RNAi technologies. “Over the course of that time, we’ve found better predictive algorithms for siRNA as well as a novel arrangement of chemical modifications in the siRNA duplex that significantly reduce off-target effects as measured by microarray and cell-based assays. The result are siRNAs called Silencer® Select siRNAs, which are chemically modified siRNAs that are up to 100-fold more potent than first- or second-generation designs. You get higher specificity siRNA performance but not at the expense of siRNA potency.”
Dr. Magdaleno notes that these improvements to the siRNA duplex have a number of benefits. “One of the most important aspects is the chemical modification that enhances the specificity of the siRNA without compromising on the potency of the siRNA,” she adds. “The combination of the chemical modification with higher potency siRNA together results in siRNAs that demonstrate coherence of cellular phenotypes between different siRNAs sequences targeting the same mRNA that hasn’t been achievable until now. The end effect will be greater confidence in RNAi experimental results.”
Dr. Magdaleno’s presentation will describe the development of the chemically modified siRNA that resulted in Silencer Select siRNA. “The methodology we used during the development is important, because it was a great learning experience. We were constantly asking questions—how do you define an off-target event? How do you reduce it? How do we know we reduced it? We have identified a combination of bases that when modified, enhances specificity, but at the moment we don’t yet understand how the modification improves specificity. We know that the reagents researchers use are critically important in their experiments. They must have confidence in the technology they are using.”