Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
RNA interference (RNAi), a powerful tool for knocking down the expression of specific genes, is widely used as a method of inhibiting protein expression. Researchers use RNAi for a range of experiments, including functional gene analysis, target validation and pathway analysis. RNAi is also a promising therapeutic vehicle. Reflecting the importance and interest in the technology, many new tools have emerged even in the seven months since Biocompare last covered this methodology. Here we discuss some of the latest developments and offerings available to researchers working with RNAi.
New tweaks on proven tools
Creative new tweaks to existing molecular tools are making RNAi products even better. Dharmacon (part of GE Healthcare), for example, offers an array of RNAi tools. The company has developed molecular tricks for better RNAi delivery, efficacy and minimizing off-target effects. For example, its ON-TARGETplus small interfering RNAs (siRNAs) offer increased specificity, and its Lincode siRNAs offer knockdown tools for long noncoding RNAs. The company’s miRIDIAN synthetic microRNA inhibitors’ patented dual-hairpin design makes inhibition more potent and longer lasting.
Dharmacon also offers a range of long-term knockdown tools using short hairpin RNAs (shRNAs) expressed in lentiviral vectors. “Our GIPZ and SMARTvector shRNA products all use a microRNA-adapted scaffold for improved processing and strand loading into RISC,” says Louise Baskin, senior product manager at Dharmacon. Both shRNAs and microRNAs also can be expressed in an inducible lentiviral vector, in which “expression of the shRNA and microRNA are under tight, dox-inducible control, allowing temporal and dose-dependent regulation,” says Baskin. The expression is controlled by “the latest generation Tet-On® 3G, which ensures minimal leakiness and potent activation upon induction,” according to Baskin.
QIAGEN offers FlexiTube and FlexiPlate siRNAs for short-term knockdown and SureSilencing shRNA Plasmids for longer-term knockdown. In addition, the company offers the “Allstars Control siRNA for siRNA transfection optimization, and as negative or positive control in experiments,” says Vikram Devgan, director and head of the Center of Excellence for Biological Research Content Business at QIAGEN. “A customization option is available for fluorescent labels and base modification of siRNA.”
Thermo Fisher Scientific offers an array of products, including the Silencer® Select siRNA systems and Stealth RNAi™ siRNA, along with mirVana™ miRNA mimics and inhibitors. Integrated DNA Technologies (IDT) includes Dicer-substrate RNAs (which have greater potency than traditional siRNAs) in its TriFECTa® kit for RNAi.
Creating new ways to enter cells
Even the best molecular tools are ineffective unless they are properly delivered into cells or animals. Thermo Fisher Scientific has a comprehensive line of Lipofectamine® reagents to introduce DNA and mRNA into cells. Lipofectamine® MessengerMAX uses a new lipid nanoparticle technology designed to maximize mRNA entry into cells; the technology is particular useful for difficult-to-transfect stem, neuronal and primary cells.
Dharmacon’s new Accell siRNAs are modified to enhance delivery into difficult-to-transfect cells without a transfection reagent, using Accell’s “patented chemical modifications,” says Baskin. This is especially helpful for using siRNAs in neurons, immune cells and stem cells, without a viral vector.
Therapeutics for the future
Pharmaceutical companies also are researching methods for delivering RNAi reagents into patients as potential therapeutic tools. One example comes from Silence Therapeutics, a Europe-based biotechnology company that uses its patented Lipoplex lipid-delivery method to introduce proprietary siRNA technology (AtuRNAi) into the liver, lung and vascular endothelium. The company’s technology has been “used in pre-clinical settings, with collaborators exploring the potential of siRNAs as a therapeutic in disease models,” such as pulmonary arterial hypertension and pre-eclampsia, says Rozi Morris, communications manager at Silence Therapeutics. “We are also in the clinic in phase 2a trials for pancreatic cancer with encouraging preliminary data,” says Morris.
Tekmira Pharmaceuticals also is developing proprietary RNAi reagents for therapeutic use. Tekmira encapsulates RNAi reagents in lipid nanoparticles (LNPs), which aids in delivery and uptake into cells. The company’s LNP delivery technology recently was licensed by Dicerna, a Massachusetts-based pharmaceutical company, for use in treating primary hyperoxaluria type 1, a liver disorder resulting in kidney failure.
A boost from the Internet
Improvements to RNAi research are being shared globally, thanks to the Internet. IDT recently developed a new site-selection tool for designing siRNAs and is expanding it for long noncoding RNAs (lncRNAs). The selection tool improves upon a previous version by employing a different kind of algorithm. “It’s a machine-learning tool that finds patterns which are beyond the ability of humans to perceive,” says Mark Behlke, IDT’s chief scientific officer. The site-selection algorithm is available now through the company’s tech-support staff and will be available on IDT’s website by summer 2015.
Addgene is a nonprofit biotechnology company that acts as a repository and distribution center for shRNA-encoding plasmids that are useful for RNAi experiments. Access through the Internet via the Addgene website enables researchers to search for available plasmids and accompanying background information. “We have almost 1,000 plasmids in the category of RNAi reagents, and this number will continue to grow, as more published and useful plasmids are being added to the library every day,” says Joanne Kamens, executive director at Addgene.
Kamens says scientists appreciate knowing the past history of their plasmids. “Since the plasmids in our repository have generally been published, or requested and used by others, the requesting scientist has some level of validation data for the reagent,” she explains. “This makes them much more valuable as research reagents. The more available data for a given plasmid, the more the scientist can trust that time spent on an experiment will be worthwhile.”
The number of requests for RNAi plasmids is slowing, Kamens adds. “We believe this is due to the rapid—explosive, really—growth for the CRISPR/Cas9 genome-engineering technology,” she says. But that needn’t be the case. “Validation of results by multiple methods is a hallmark of good experimentation,” she says, “and scientists will continue to use RNAi reagents as a productive method to validate hypotheses.” Behlke agrees that it’s best to “use both RNAi and CRISPR to cross-validate each other. It’s an extraordinarily powerful way to avoid being [led] down the wrong path.” Indeed, the continuing proliferation of RNAi tools indicates the technique isn’t likely to fade anytime soon.
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