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RNAi Meets Microarrays: Uses Range from Sequence Selection to SARS Drugs

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It seems like one of those fated celebrity pairings: the still-simmering microarray meets RNAi, the new darling of functional genomics, and the RNAi microarray is born. But the reality is still in the experimental stages, and it’s not yet clear how this marriage will turn out.

RNAi, or RNA interference, involves transfecting a cell with a double-stranded sequence of RNA, triggering a process that silences expression of corresponding genes in the cell. RNAi works one gene and one cell at a time, but RNAi microarrays promise to make these assays high-throughput.

“There’s a few advantages” to doing RNAi on microarrays, said Spyro Mousses, who is experimenting with this technology in his work as director of the cancer drug development laboratory at the Translational Genomics Research Institute. “The first is it allows you to miniaturize both the transfection and the assay, and the reason that’s important is you use less reagents and less [RNA] ... the second is, it allows you to have uniformity, [and] the third is because the spots are close together, you can use various scanning technologies to analyze the samples [more] than you can on any other platform.”

At the May RNAi - 2003 meeting in Waltham, Mass., Mousses gave a talk on his proof-of-concept studies of RNAi microarrays for studying gene function in cancer cells. First, he said, he conjugates short sequences of interfering RNAs (siRNAs) to a cationic lipid, and uses a standard microarraying robot to print these onto glass slides, in 200 µm spots that are spaced 300 µm apart from one another. Then, adapting a method invented by Whitehead researcher David Saba tini, Mousses places the microarray in live cell culture. “An adherent monolayer of live cells forms on the microarray,” he explained in his talk. The siRNAs enter the cells, with the lipid serving as a vector for transfection.

In an initial study, Mousses spotted a slide with RNAi strands known to suppress the gene that encodes green fluorescent protein, then covered the slide with HeLa cells that were engineered to express GFP. In this fluorescent green lawn of cells, the areas around the RNAi strands became black holes, indicating that the expression of GFP had been silenced.

Mousses is already using this cell microarray technology to silence genes in other cell lines. He is also working toward an ultimate goal of making a validated genome-wide library of siRNAs.

Meanwhile, Vivek Mittal, of Cold Spring Harbor Laboratory, has used this same cell array technology to test out siRNAs for efficacy. Mittal has spotted up to 200 siRNA spots on a glass slide, and also printed a plasmid that expresses the proteins, which are combinations of a GFP and the protein encoded by the gene targeted for silencing. He adds red fluorescent proteins as controls. On top of this, the lawn of cells is placed. “Inhibiton of GFP will tell you what is working the best,” Mittal said.

RNAi microarrays are particularly useful for validation, Mittal said, due to the difficulty of otherwise finding an siRNA that will knock out a gene. Mittal’s group was looking for siRNA to suppress expression of the ID genes, which are involved in tumor angiogenesis, and “we were extremely unlucky. You can design siRNA probes, against a gene..there are some rules, but they are not very robust rules. Even if you design an siRNA using these rules, there is no guarantee they work.” Mittal has had some cases where his group designed eight siRNA probes against a gene, and found no hits.

With the RNAi microarrays, Mittal has also found that testing for gene suppression is faster than with other methods, such as Western blots using antibodies. Since he looks at ectopic suppression of the gene, not endogenous suppression, it takes as little as 24 hours to tell whether a sequence works. The group has tested the technology on both human and mouse cells.

Speed is also a key advantage for CombiMatrix scientists — the only commercial group to so far make use of RNAi microarrays. But instead of using the microarray as a tool for gene suppression using RNAi, the company has used its electronically labeled microarray probes as building sites for siRNAs — initially as potential therapeutics against SARS. “The chip is essentially a little siRNA factory,” CombiMatrix CEO Amit Kumar said.

CombiMatrix’s arrays consist of a semiconductor coated with a three-dimensional layer of porous material in which DNA, RNA, peptides, or other small molecules can be synthesized or immobilized within discrete test sites. The company’s proprietary software directs sequence synthesis one base at a time, using individually controlled electrodes on the surface of the semiconductor.

Recently, the company had adapted this technology for use in SARS genomics, launching in late April a custom chip for different variants of the SARS coronavirus. Using the microarray, company scientists started on the RNAi path after identifying two genes that they thought were critical for replication of the virus, said Kumar. “We wanted to inhibit these two genes, and we looked at all these various siRNA sequences using standard siRNA design criteria. On top of that, we used the added capability of our software, which included Blasting these sequences against the human genome to make sure that there was little orthogonality.” The scientists emerged with 60 siRNA sequences that they believed were good candidates for stopping the SARS coronavirus in its tracks.

Next, the team at CombiMatrix built precursors to these sequences on their chips, along with promoters that would allow them to amplify and transcribe the sequences. They built the sense and antisense strands of the siRNA separately, as these strands would cleave together once pooled. “This is really the first chip-based approach to making siRNAs,” Kumar said. “It took us a few hours to do. Ordinarily you would have to synthesize each sequence off line using a chemical synthesizer. It would take you weeks.”

CombiMatrix has since shipped these siRNAs off to SAMRID, the US Army Medical Research Institute of Infections Diseases, which is testing the coronavirus, Kumar said.

The company is planning to develop, and synthesize on its chips, other siRNAs against viral and bacterial pathogens, including possibly HIV, HCV, West Nile virus, and Anthrax. Kumar estimates that the company can identify the siRNAs for a particular pathogen, and synthesize them. The company is in discussion with several potential partners for this project, said Kumar.

However, “an siRNA that does work is still a long way away from an actual drug,” said Kumar. “You still have to go through preclinical and human clinical testing. That being said, we’re pretty confident that one of those 60 will work.”

Even with these early successes, RNAi microarrays require a lot more development, especially, in the case of the cell arrays, the protocols and the analytical tools. RNAi microarray technology “is in its infancy,” said Mousses.

— MMJ

This article originally appeared in BioArray News.

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