As the use of RNAi agents for research becomes more commonplace, and the promise of RNAi as a therapeutic modality moves closer to reality, some researchers have been looking for siRNA applications that go beyond gene silencing.
One of these researchers, University of York professor Jo Milner, has developed a nuclease-resistant siRNA termed a "crook" siRNA for its resemblance to a shepherd's crook that knocks down expression of a target gene and primes PCR amplification for its own quantification. Details about the crook siRNAs were recently published in Nucleic Acids Research.
"The problem with siRNA is its short life following systemic administration," Milner told RNAi News this week. Thus, "the motivation for this [work] was to see if we could produce [an siRNA] that had resistance to nuclease digestion, [but] retained the ability to induce RNA interference, and … could be detected at the very low levels that are all that is necessary to induce RNA interference."
"The problem with siRNA is its short life following systemic administration. The motivation for this [work] was to see if we could produce [an siRNA] that had resistance to nuclease digestion, [but] retained the ability to induce RNA interference, and … could be detected at the very low levels that are all that is necessary to induce RNA interference."
To do so, Milner and her colleagues modified the 3' end of an siRNA's sense strand by adding a 19-nucleotide-long DNA sequence designed to allow the siRNA to function as a PCR primer and form a stable hairpin structure. This DNA loop, she said, has previously been demonstrated to confer stability, and yet "it's not too stable, so you can do PCR amplification of that extended DNA" sequence, she said. "Essentially, we were taking information that was already demonstrated to construct a novel siRNA."
To test their approach, the researchers used a line of human cervical cancer cells positive for human papilloma virus type 16 as a model in which to compare the effects of crook siRNAs and unmodified siRNAs, both targeting the viral E7 gene.
They first examined the ability of the crook siRNA to act as a PCR primer. "We … altered the standard PCR protocol to allow for a limiting quantity of the E7 crook siRNA (upstream) primer," the researchers wrote in the Nucleic Acids Research paper. "Thus the E7 crook siRNA primer is limiting for the reaction, whereas the second (downstream) primer and the DNA template are added in excess for the reaction.
The crook siRNAs were found to support PCR and, "as predicted, the product yield tended to plateau at lower levels with increasing dilution of the E7 crook siRNA primer," the researchers wrote in the paper.
"We've been able to detect the crook siRNA down to attomolar levels per cell," Milner said. She noted that while there has been a report of cellular detection of similar levels of siRNA by researchers using liquid hybridization with a P-labeled probe followed by a nuclease protection step, this "requires a radioactive approach and is quite complicated. It [also] might be affected by different sequences of the siRNA," she noted.
With crook siRNAs, "we have a system in which you can slot any siRNA sequence you want, and the DNA extension is constant," Milner said. "That's the bit that the PCR reaction is dependant upon … and therefore we shouldn't get variations due to different [RNA] sequences during the quantitation reaction."
In their experiments, Milner and her colleagues also found that the crook siRNAs are as efficient in knocking down their targets as their unmodified counterparts. Additionally, "the crook siRNA is not intrinsically toxic to cells in culture," she noted.
"Given the similarities between crook siRNA and unmodified siRNA for the induction of RNAi, we anticipate that simple PCR-based detection and quantification of crook siRNA will prove valuable in the development of RNAi therapeutics and informative on siRNA cellular uptake, intracellular localization, tissue distribution, and pharmacokinetics," Milner and her colleagues wrote in the Nucleic Acids Research paper. "However, the applications of this new technology are not restricted to therapeutic development, or even to mammalian systems. We anticipate that PCR-based detection of siRNAs, such as crook siRNA, will prove a powerful adjunct for RNAi-based studies in general."
"One thing that we'd like to develop, and if we don't, hopefully others will, is in situ PCR" using the crook siRNA, Milner noted. "At the moment, to detect siRNA delivered into cells often [requires] a fluorescence tag … [and] far greater levels of the siRNA molecules than [are] required with this PCR-based method."
With an in situ method, "when you give a dose of this crook siRNA into an animal, say, you can then … monitor its distribution within various tissues … [as well as] the stability and excretion routes," she said.
At the moment, Milner has not conducted any in vivo experiments with the crook siRNAs. "My main interest is more in using RNA interference to study apoptosis in cells," she said. "This methodology was an idea that we [explored to see] if it would work. Hopefully, [now that] we've shown it will work, others will come in … and develop it for animal experiments."
Doug Macron ([email protected])