Researchers at the Molecular Sciences Institute in Berkeley, Calif., have come up with a way to detect very low-abundance proteins by using tadpole-shaped protein-DNA chimeras which can be amplified using PCR.
According to Roger Brent, the director of the laboratory where the studies were conducted, the protein-DNA tadpoles can offer a limit of detection that is 109 lower than the traditional ELISA approach.
“Mass spectrometry methods can’t easily detect low-abundance stuff,” said Brent, whose study, led by research fellow Ian Burbulis, is published in the January issue of Nature Methods. “With this method, we could detect as few as 600 molecules with 95-percent confidence.”
The tadpole tool consists of a “head” made up of a protein element, such as an antibody, that has targeted affinity for a specific molecule. The “tail” of the tadpole consists of a DNA tag that enables PCR-based quantification.
The tail and head are joined by exploiting the natural phenomena of inteins — peptides that excise themselves from a polypeptide chain. Once the intein excises itself, it leaves a peptide with a C-terminal end that would naturally join up with a cysteine amino acid, Brent explained. But instead of the cysteine, the peptide is “tricked” into binding with a DNA attached to a cysteine-like moiety.
“You need to be able to do standard recombinant DNA techniques with facility, and to purify proteins in order to synthesize these tadpoles,” said Brent. “If you’re doing it yourself in your lab then the incremental cost in terms of reagents is in the hundreds of dollars [for one tadpole].”
Brent said he is open to collaborating with companies to produce commercial tadpoles.
“In the future, every researcher should be able to order up a pretty antibody molecule against any protein they care about,” said Brent. “The tadpole would be the next step beyond it.”
Brent said he envisions applying the tadpoles to clinical settings, environmental studies and research in general.
One application would to be make tadpoles against proteins that are found through mass spectrometry to differ in expression level in normal versus diseased states.
“We envision taking the mass spec signature and making something more sensitive that could be used to quantify the proteins inside human serum, for example,” said Brent. “You can’t detect rare things very well with a mass spec. It isn’t as sensitive of a detection method.”
The idea of coupling a DNA tag to an antibody is not new, said Brent. In fact, the first paper that coupled DNA to protein came out in the early 1990’s. However, there were flaws with the molecule that prevented it from being used on a wider basis for detection of low-abundance molecules.
“Here, you know what you’re getting. There’s no glycosylation, and we’re in control of the biochemistry,” said Brent.
In an article that accompanied Brent’s research paper, Stanford microbiology and immunology researcher Garry Nolan assessed the tadpole and concluded that its simplicity, adaptability, and sensitivity “make this an appealing system for researchers wanting a standardized, high-throughput and accurate detection system for just about anything.”
Nolan pointed out that when DNA tails were fused with aptamers — short peptide sequences with only modest affinities for targeted molecules — the tadpoles performed with as much sensitivity as high-affinity antibodies in ELISA assays. This equivalent sensitivity using aptamers is significant because aptamers are much cheaper and easier to make than high-affinity antibodies, Nolan said.
Tadpoles are not limited to proteins, Nolan said. The DNA moieties could be coupled to proteins that bind a vast range of molecular entities.
“At the front end of the tadpole, one can envision using nearly any encodable natural, designed, or evolved protein domain that can bind numerous target classes, ranging from small organic molecules or ions, to protein fragments in the plasma of patients with early-stage cancer, protein-protein contacts, or proteins produced in early-stage viral or biological infection,” said Nolan.
In terms of environmental monitoring, Brent said he envisions using tadpoles to monitor, for example, 200 liters of air in an airport for smallpox virus coat protein, or to determine where a contaminant in lipstick is coming from within a lipstick factory.
In terms of cellular applications, Brent’s research group is working on using tadpoles to compare how much of a protein is present 30 minutes after stimulation versus 50 minutes after stimulation.
“We’re pushing using tadpoles for detection and counting of small numbers of molecules within cells,” said Brent.
In order to make tadpoles widely accessible, the MSI researchers need to streamline their synthesis, Brent said. One improvement that could be made in the synthesis process is to use bacteriophage-derived antibodies instead of antibodies synthesized by mice and rabbits.
“Using modern recombinant techniques with bacteriophages and yeast, it should take weeks instead of taking a month and a half to make an antibody,” said Brent. “It’s time to free mouse-kind and bunny-kind from their long bondage to humans.”