New Method for Detecting Point Mutations Could Significantly Impact Low-Cost Diagnostics

NEW YORK – A new method of determining point mutations associated with infectious disease drug resistance could provide a path to cheap diagnostics for low-resource regions.

In a study published in Cell last month, a team led by researchers at the Biodesign Center for Molecular Design and Biomimetics at the Biodesign Institute at Arizona State University developed programmable riboregulators (SNIPRs) as a method to detect single-nucleotide mutations in genes. 

The SNIPRs are messenger RNAs that, when initially synthesized, fold into a secondary structure that prevents translation of a reporter gene, according to Alexander Green, a researcher at the center and the study's lead author. The SNIPR conceals the site that the ribosome binds to before starting translation, and when the SNIPR detects the target RNA it will hybridize to that target and unfold. If the SNIPR encounters unmutated RNA there is a mismatch and protein translation is blocked. 

In the study, the researchers observed a 100-fold difference in gene expression between the mutated and unmutated RNA using E. coli bacteria. 

The SNIPRs are designed to search for specific mutations by Green and his lab using software that generates "the optimal RNA sequence that will match up to the particular target to enable it to be detected," Green said.

In other words, the system works like a seesaw. "You set up the system such that it's perfectly balanced so that the sides of the seesaw match one another, everything's at equilibrium," Green said. "If you have a point mutation … this change tilts the seesaw in the opposite direction, which makes the reaction unfavorable and stops the detection reaction from going forward."

One of the benefits of this technique is that it can be applied in multiple ways, either in living cells or as a paper-based diagnostic. If someone knows a particular mutation leads to drug resistance, the SNIPRs can pinpoint that mutation and determine if a parasite is resistant to that drug, Green said. They can also use wild-type sequences to determine if a sequence is off or has a mutation that could cause problems.

In the study, SNIPRs were developed for colorimetric detection of multiple clinically relevant mutations, including two mutations that occur to the RT, M184V, and K65R genes conferring NRTI resistance for HIV and the dominant mutation for resistance to artemisinin, the C580Y mutation of the K13 propeller domain of the parasite. The team also developed SNIPRs against the mutations of BRCA1 P871L, BRCA2 N372H, BRCA1 185delAG, and BRCA1 5382insC, which increase the risk of breast and ovarian cancers.

Green said he had the idea for this method in 2016 while preparing a grant submission to the Gates Foundation for solutions for detecting drug-resistant tuberculosis and malaria. It then took a year to prove the concept, and "another year or two to get all the testing done and demonstrate that it works pretty well against a lot of different target sequences," Green noted.

The Gates Foundation ended up funding the early initial development phase of the project, with additional funding provided by agencies including the National Institutes of Health and the Arizona Biomedical Research Commission.

SNIPRs can be applied to isothermally amplified DNA or RNA to analyze mutations. The mutated targets cause a color change in paper-based cell-free assays at 37° C, revealing whether the sample has that mutation. If the assay remains yellow, the sample is in the clear, but a change to purple signifies the presence of a mutation.

These tests can also be performed at the point of care with very little instrumentation, allowing for creative applications. Because the reaction occurs at 37° C, the user would only need a hot plate, or could even strap the paper to someone's armpit to measure the results out, Green said.

Green noted that the technology could be particularly useful in low-resource settings, where access to a centralized lab isn't possible.

That ability to move outside of the lab is what Keith Pardee, an assistant professor at the University of Toronto who has worked with Green on paper-based nucleic acid tests, finds particularly exciting about the study.

"It basically brings the capacity to genotype out of the lab potentially and into the hands" of regular people, Pardee said. But getting the technology into the hands of users is also one of the biggest challenges for synthetic biology-based sensors, Pardee noted. "It can certainly be run by someone with some skills to use a pipette," Pardee said. "But the next challenge is how you place it robustly into the hands of users, say frontline healthcare workers."

Pardee declined to go into specifics, but said he's working to submit a paper using personal diagnostic devices to interface with the sensors.

Although SNIPR applications for infectious diseases are the main focus of the study, Green said he also sees the potential for SNIPRs to be used for patient self-testing for genetic mutations. "If you know your mother has particular mutations that could lead to cancer, this can be a quick way to detect whether you have the same mutations," Green said. "You wouldn't need to go through the full sequencing process to verify that."

Another plus offered by this self-testing, Green added, is the safety of patient data. "If you do it on your own, you're not sending your DNA to some company," Green said.

SNIPRs can also be used to determine how drug-resistant microbes spread and contain them before they reach areas that can't deal with drug resistance, Green added.

In addition, SNIPRs, which are part of the broader field of paper-based nucleic acid tests, could possibly be used in pandemic situations such as the current SARS-CoV-2 outbreak, Pardee added.

"I think there's a change to add a lot of surge capacity to the current RT-qPCR-based diagnostics," Pardee said. "In environments like rural areas, northern communities in Canada and low- and middle-income countries, where PCR is not as available, the potential for this category of diagnostics is to fill that gap."

Beyond human health, the method could also be applied in other industries, such as agriculture for tracking pests or pathogens, national security for determining bio-threats, and food safety and water quality measures, Pardee said. 

The next steps for Green and his team are testing the SNIPRs on larger numbers of samples to validate the tests more fully, both for the drug-resistance testing and human genotype testing, which he said will take probably a year or two. Eventually, however, they'd like to bring the test to the market, either via a spinoff company or a partnership with an existing diagnostic firm – although Green noted that those decisions would be several years down the line. Green and his co-authors Fan Hong and Hao Yan have also filed patents for the SNIPR method.

"We think they could be really helpful in diagnosing different diseases or identifying the best therapeutics that a person should take if they're infected with a particular pathogen," Green said.