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Scripps Team's Self-Replicating RNA Enzymes Quantitatively Analyze Proteins, Other Targets

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A team led by the Scripps Research Institute's Gerald Joyce is beginning its second year of an effort to develop a process analogous to quantitative PCR, in which the growth rate of a synthetic RNA molecule can be used as a quantitative measure of the initial concentration of a target protein or other analyte.

Joyce and his team received $324,000 this month from the National Institutes of Health to support the second year of a four-year project to develop the system, based on autocatalytic aptazymes: RNA enzymes that undergo self-sustained replication with exponential growth at a constant temperature, initiated and constrained by the concentration of a particular ligand or target molecule in a sample.

"Fundamentally the reason PCR is so cool is that it goes exponential. That's why it's so powerful, that’s why you can do qPCR, because the exponential growth rate — the number of cycles before you hit threshold — is a nice readout of the concentration of the analyte," Joyce told PCR Insider.

"So it's great, but it's great only if what you are trying to measure is a target nucleic acid, [not a protein or a drug or a metabolite], " he said.

According to Joyce, the group started its project several years ago with the development of the self-replicating RNA enzymes. "We made a molecule out of RNA that self-replicates – it just goes nuts," Joyce said.

Then the team engineered them to operate as aptazymes, which depend on the presence of a ligand to undergo their exponential amplification.

However, these first aptazymes had limited clinical appeal because the biological RNA they were made up of is quickly broken down in clinical samples by native ribonucleases. These can be removed from a sample, but only through processes that scrub the sample of all proteins, which would be a non-starter if the clinical target is itself a protein, Joyce said.

"The biggest flaw of this thing until this year was that no one wants to use RNA to recognize things because of how fragile it is. If you put RNA in human serum, it's degraded instantly, so within a couple minutes it's gone," Joyce said. "We could measure analytes in serum, by first deproteinizing and then doing the measurement. But if you want to measure a protein, you can't do that."

To solve this problem, the team decided to take a synthetic approach, reconstructing the aptazymes as their enantiomeric twins — essentially mirror images. These non-biological, L-RNA molecules have the same autocatalytic properties but are completely resistant to breakdown by ribonucleases.

"RNA, like proteins, is handed. It's all D-sugars," Joyce explained. "But you can make an RNA molecule that’s the mirror image of biological RNA. It's like RNA from another planet," he said. "The nucleases can't touch it."

The group published a report this May in the Journal of the American Chemical Society describing the synthetic approach and initial tests of its performance, showing that the L-RNA reaction system undergoes the same isothermal exponential amplification as the natural D-RNA system, but can operate in the presence of human serum.

According to Joyce, the group believes the approach has clinical potential for multiplex, quantitative detection of protein or other small-molecule targets in biological or environmental samples, especially with the problem of RNA degradation now taken care of.

"I don't see this as a startup kind of thing … I don't want to call it a parlor trick, but it is a molecular trick. So one would want to, I think, piggyback this on an existing fluorescent readout system," Joyce said. "Plenty of companies out there that have the right hardware to go with it. We just have to make the molecular software better."

As the group moves forward under its second year of funding, and beyond, Joyce said the lab will focus on refining the new L-RNA-based enzymes using directed evolution so that they operate more efficiently.

"We are trying to just make this faster. Now, it takes about an hour to get to threshold, whereas with qPCR people don't even want to wait that long," Joyce said.

The team will also focus on making the system more generalized, so that the enzymes can be easily combined with different aptamers for a wide range of target molecules.

"What you'd really like to do is be able to make one of these [that is adaptable] for all the circulating human proteins," he said. "And we don't want that to be as tedious as what it would take to raise a high-specificity, high-sensitivity antibody for all of them."

According to Joyce, making aptamers has become relatively routine. "What's not yet automatic is to guarantee that the aptamer is going to function in the context of this replicator with the sensitivity and specificity we like," he said.

"We want to make that process much more bullet-proof and automatic so you could take any aptamer out there and plug and play."

As part of the project, the researchers also plan to integrate real-time, multiplex fluorescent readout into the process. In its experiments, Joyce said the group has been using a commercial qPCR machine, a Bio-Rad CFX, tricking it into running at a single temperature rather than thermal cycling.

"I like [the CFX because it has lots of different channels for different wavelengths – so you can play multiplex," said Joyce. "We have populations of these replicators that compete [against] each other and evolve — you can have thousands, each with different recognition sequences, reproduce at the same time."

"The CFX can measure six wavelengths so we can have six replicators measuring six different [targets] at a time in each well," he said.

Joyce said the system, if eventually commercialized, would most likely offer an alternative for some, but definitely not all, ELISA, applications. "For sensitivity, specificity, multiplex, this could do some things ELISA can't do," he said.

Alternatives that try to marry PCR with antibody-based techniques, like immuno-PCR, have not caught on, and do not offer the potential of quantitative analysis, Joyce added. "You have to raise the antibody for everything you're after. That's challenging. Then the antibody, especially for metabolites, doesn’t always have the best affinity."

"To do quantitative immuno-PCR would be really hard."

Joyce said the group believes the NIH support for the project reflects its ability to potentially fill this clinical niche. "They recognize this for [that fact] — that there are very few things like this that go exponential," he said.

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