NEW YORK (GenomeWeb) – A researcher at the University of Maryland has been awarded a two-year grant from the National Institute of Allergy and Infectious Diseases to develop an aptamer-based HIV detection assay.
The assay detects binding between the reverse transcriptase protein of HIV, or HIV RT, and a DNA-based aptamer developed in the lab.
Molecular geneticist Jeffrey DeStefano told GenomeWeb the new funding, which amounts to about $50,000 per year, will push forward ongoing work in his lab on aptamers and HIV. His group had previously used systematic evolution of ligands by exponential enrichment, or SELEX, to zero in on the primer-template sequences that HIV RT most preferred. They published a detailed description of a 38-nucleotide HIV RT-binding DNA aptamer in Nucleic Acid Therapeutics in 2012.
In the current iteration, the aptamers now have "some O-methyl additions to specific nucleotides," DeStefano said, adding, "These now bind [HIV RT] extremely tightly … they have [dissociation constants] around 20 or 30 femotomolar, and have half-lives in the 100 minute range," he said.
Recently researchers have gotten around sensitivity issues with PCR-based HIV detection using droplet digital PCR, and the high cost of HIV screening by using pooled nucleic acid testing in the clinic. But the aptamer-based assay is inherently sensitive, quantitative, and will likely be inexpensive.
"[PCR is] a great technique, obviously, it's changing the world, but there are issues with time, contamination, and false positives that this assay would tend not to have as much of; I'm not saying it's a better assay by any means, I'm just saying I think it's a really reasonable alternative method," DeStefano said.
The method is already more sensitive and specific than standard rapid antibody-based testing for HIV. At this point, it takes about as long as a typical lab-based PCR assay, DeStefano said. However, he hopes the new funds will allow the lab to focus on speeding up the reaction. It can now detect about 100 particles of HIV virus, which means it might be useful even for latent, late-stage infections. It could also be useful for HIV laboratory research, and possibly adapted to detect other retroviruses or pararetroviruses that depend on RTs.
The aptamer happens to mimic a segment of viral genomic material that is crucial for its life cycle and contains a purine-rich domain, known as the polypurine tract, DeStefano said. "The [RT] really likes things that look like the polypurine tract ... it basically binds really tightly to them such that you can bind the enzyme and wash away everything else," he explained.
Interestingly, DeStefano has also been collaborating to use so-called unnatural base pairs to build aptamers. With Phil Holliger, a researcher at the Medical Research Council in the UK who recently reported in Nature on catalytic activity of xeno-nucleic acids, or XNAs, DeStafano has made aptamers that also bind extremely tightly to HIV RT. However, one disadvantage at the moment is that these aptamers can't be extended, "so it's harder to detect that RT is there," DeStefano said.
As reported by GenomeWeb, a team at Scripps is one of a handful of labs at the forefront of creating high-fidelity XNAs, and that lab recently showed XNA incorporated into E. coli could replicate in living bacteria. XNA aptamers have been catching on, DeStefano noted, in part because usual immune system and cellular mechanisms do not target them for degradation.
The lab is also collaborating with Indira Hewlett, an HIV researcher at the US Food and Drug Administration, with the hopes of further developing aptamer-based detection methods of HIV "that can potentially be used in the field and are accurate, reliable, [and] stable," DeStefano said.
The current assay is performed in a 96-well plate with RT-driven aptamer extension using radiolabeled nucleotides. But DeStefano envisions it could potentially be built into a colorometric assay. The RT itself is extremely stable, as is the aptamer, and this may make the method amenable to building an HIV diagnostic, possibly one useful in low-resource settings.
Roche currently markets a colorometric assay that detects enzymatic activity of reverse transcriptase and is available for research use only. DeStefano was unaware of any entities developing assays for HIV using aptamer-based detection of RT specifically.
The lab has filed an intellectual property disclosure with the university describing the method as well, DeStefano noted. However, for later stages of development, he said he would likely seek industry partners. "They have tools, and even ideas, that we don't," he said.