While a number of technologies are being developed to turn the RNAi process on and off, these typically require introducing small molecules or other compounds into the cell. But a researcher from Louisiana State University, however, thinks that a photoactivation technique developed three decades ago may offer a better solution.
The approach, called photocaging, involves covalently attaching blocking groups onto a nucleic acid. These groups keep inert the nucleic acid — an siRNA, for instance —while protecting it from enzymatic degradation until it is exposed to light, at which point the nucleic acid’s activity is restored, according to Todd Monroe, an assistant professor in LSU’s department of biological and agricultural engineering.
“There are a lot of small molecules that can drive gene expression or gene repression, but those molecules, if they are not already inherent in some type of pathway, have to be delivered” and can be difficult to regulate, he told RNAi News this week.
With photocaging, on the other hand, researchers can control when and where in a cell a nucleic acid is activated, he said. “If we’re working with an organism instead of a culture dish, we can deliver [a caged nucleic acid] in a select spot” such as the retina.
According to Monroe, photocaging was developed in the 1970s to inactivate and reactivate ATP to study the kinetics of muscle contraction. “We’d like to take those same principles [used with ATP] and apply them in cells and tissues to govern nucleic acid bioactivity,” he said.
Monroe and his colleagues have been working on this concept for some time and have conducted experiments in which they attached nitro benzyl-like cage compounds to the phosphate backbone of DNA. This caging process “disrupts transcriptional enzyme machinery [and] blocks [the DNA’s] degradation by nucleases,” he explained. When exposed to light, the molecules reactivated.
“Then we moved to caged oligos and have shown … that you can block their hybridization with a complementary strand,” Monroe said. “That’s what [led to] the idea to move towards RNA interference. We thought if we could do this with an siRNA — protect it from enzymatic degradation and block its interaction with the RISC machinery or … a complementary mRNA target in the cell — we’ll effectively inactivate it until we go in there with light and turn it back on to let the normal RNAi process resume.”
Last year, a research team from the University of Bonn in Germany published a report showing that they can use caging to inactivate siRNA and use light to reactivate it. However, Monroe noted that without significant modification, photocaged siRNAs are often unstable.
“We thought if we could do this with an siRNA — protect it from enzymatic degradation and block its interaction with the RISC machinery or … a complementary mRNA target in the cell — we’ll effectively inactivate it until we go in there with light and turn it back on to let the normal RNAi process resume.”
The caging compound they use — 1-(4,5-dimethoxy-2-nitrophenyl)ethyl, or DMNPE — can be attached to the phosphate group of a traditional siRNA, but this makes the oligo susceptible to self-catalyzed hydrolysis, he said. However, “we developed a fully-2’-fluorinated siRNA that we call siFNA, or small-interfering fluoronucleic acid … [that] actually participate in the RNA interference pathway” without self destructing.
In October, Monroe and collaborators from LSU published data in Chemical Biology and Drug Design showing that these siFNAs could induce RNAi and with silencing activity nearly equal to unmodified siRNAs. The fully-2’-fluorinated nucleic acids products also demonstrated “superior resistance to digestion over native RNA,” the paper’s authors wrote.
With these data, as well as a five-year, $400,000 National Science Foundation grant, Monroe is beginning additional work on optimizing and testing the photocaged siFNAs.
Part of that work involves the development of new cage compounds “so that we have the ability to put these into an oligo at several different locations,” he said. “We’d also like to work with some of the cage chemistry to be able to attach [the compounds] to native or unmodified RNA.”
To make that work, he said he will need to develop “some new and better cage compounds, and means to attach these to specific sites along an RNA molecule during synthesis.”
These first experiments will examine the ability to inactivate and reactivate reporter genes in culture to demonstrate proof of concept, Monroe said, after which he plans to move the work into zebrafish.
“In the later stages of the project, we’d like to pick up more clinically relevant genes — say, ones that are key directors in a developmental process — and be able to silence those at a specific time and place in a developing [zebrafish] embryo to see what happens to the surrounding cells and the organism as a whole,” he said.
Additionally, the zebrafish work will help determine tolerance thresholds for the activating light, which Monroe said can have some cellular effects.
Monroe said that over the term of the grant he expects his work to help in the development of new tools for functional genomics and developmental studies.
“However, downstream I do see clinical applications for this if we need to, let’s say, target something in the back of the eye or another optically available tissue,” he said. “I’ve even seen some folks [using] fiber optic catheters to photoactivate in the lumen of an organ, so there are aspects of [this technology for] targeted therapies if we wanted to have an RNA interference drug but needed to have tight control over spatial patterning.”