RNAi: Five Tips to Better Silencing

1. MAKE SURE YOU HAVE THE RIGHT SEQUENCE

Because RNA interference (RNAi) can vary greatly, most suppliers recommend screening four or five siRNAs per transcript. In a 2004 report, siRNA vendor Dharmacon identified eight criteria for optimal inhibitor design.¹ In general, siRNAs should be 19 to 21 nucleotides long, lack internal repeats or a high degree of secondary structure, have low (under 50%) G/C content, and lack sequence homology to off-target genes. Thermodynamic stability is also key. The study showed enhanced potency in siRNAs with low internal stability at the sense strand 3' end.

A number of free siRNA-design programs are available online from commercial RNAi tool vendors and academic­ labs.

2. START WITH NAKED siRNA

If you're just starting out, it makes sense to use "naked" siRNAs (i.e., purified double- stranded RNAs) that are delivered by transient transfection. "siRNAs make a lot of sense to use in the instances where you want a very transient decrease in expression to either test a particular hypothesis or ? to evaluate the role of a given sequence as a therapeutic," says Beverly Davidson, a neuroscientist at the University of Iowa, who uses RNAi to knock down genes involved in neurogenetic diseases.

The fastest (albeit more expensive) approach is to order siRNAs custom-made from an oligo supplier. The major RNAi companies sell annealed siRNAs in several purity formats ranging from standard (for in vitro work) to HPLC or PAGE-purified (for in vivo work). You can also make your own siRNA; commercial kits can transcribe two complementary siRNA strands in vitro for subsequent annealing and transfection. (See tables below)

3. MAKE THE RIGHT MODIFICATIONS

Some researchers chemically modify their siRNAs for greater stability or tissue targeting. Chandra Vargeese, vice president of chemistry at San Francisco-based Sirna Therapeutics, says the company's scientists have developed a portfolio of chemical modifications to 2'-OH groups to facilitate local and systemic siRNA delivery, and in some cases even modulate immunostimulatory side effects. "We can go from no modifications to fully modified siRNAs, and anything in between," she says.

Recently the company demonstrated that replacement of ribose sugar 2'-hydroxyl groups with 2'-fluorine, 2'-O-methyl, and 2'-deoxy sugars, and the use of backbone phosphorothioate linkages and 5' and 3' inverted abasic end caps, produced a thousand- fold enhancement in stability in human serum.² Modifications can significantly increase the cost of your oligos, however. Qiagen, for instance, sells cholesterol- modified RNAs for roughly twice the cost of purified, nonmodified siRNAs, according to company spokesperson Eric Lader.

4. OPTIMIZE SILENCING WITH PLASMIDS

Longer-lasting effects can also be achieved by expressing plasmid-encoded short hairpin RNAs (shRNAs) or microRNAs­ from pol III or pol II promoter sequences, respectively, in vivo or in culture.^3,4 shRNAs typically contain stem structures between 19 and 29 nucleotides in length, capped with a five-to-nine base loop sequence; microRNAs are longer, more complicated structures. Because they are transcribed using Pol II, microRNA-based constructs can be expressed conditionally (e.g., using a regulatable promoter), allowing for more sophisticated knockdown experiments. Ultimately, though, both shRNAs and microRNAs are processed in vivo into double-stranded products capable of programming the RISC.⁵

5. USE VIRUSES FOR IN VIVO WORK

Naked siRNA and shRNA expressed by a standard plasmid vector are fine for screening in cultured cells, but they don't work well in live animals. But viral vectors do. Says Davidson, "If you put naked DNA into a lung or brain you may get very little of the RNAi expressed, but you can use a virus to deliver the payload to your cells of interest or whatever organ you're targeting."

Researchers who use viral vectors ? both adenoviral and lentiviral systems are available ? can employ selectable markers to look for cells in which shRNA has been expressed, and then follow up with an assay for the desired phenotype, says David Root of the Broad Institute at the Massachusetts Institute of Technology. The RNAi effect can then be turned on or off with a conditional expression system, such as the doxycycline- or tetracycline-regulated systems from Clontech, Stratagene, and Invitrogen, among others. (see Table 2 below)

aconstans@the-scientist.com

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