SNIPRs take aim at disease-related mutations


February 27, 2020

A typo appearing in the draft of a novel is no great calamity. Nature, however, is often less forgiving of errors. A change in just one letter of the genetic code can have catastrophic consequences for human health.

Such genomic gaffes, involving a single base in a length of DNA or RNA, are known as point mutations. They can result in mild abnormalities like color blindness as well as serious diseases, including neurofibromatosis, sickle-cell anemia, certain forms of cancer and Tay–Sachs disease. Mutations can also produce disease variants that are resistant to conventional treatment.  Cell SNIPRs (for Single-Nucleotide-Specific Programmable Riboregulators), have the capacity to identify any RNA sequence based on a single nucleotide difference. Graphic by Shireen Dooling for the cover of CELL. Download Full Image

Researchers would like to detect these point mutations to better assess vulnerabilities in human health, provide accurate early diagnosis and guide appropriate therapy. Until now, however, registering subtle alterations like point mutations occurring within living cells has been challenging.

In a new study, lead author Alex Green, a researcher at Arizona State University's Biodesign Center for Molecular Design and Biomimetics and his colleagues describe a new method for detecting point mutations. The technique can be applied in living cells, offering a rapid, highly accurate and inexpensive means of identifying mutations relevant to human health.

The method can be used in conjunction with paper-based diagnostic tests (developed by Green and his colleagues), capable of pinpointing mutations and displaying a color-based readout in reactions powered by human body heat.

“What we've done with our technology is to develop a new, portable way to detect very minute sequence differences between RNAs you're trying to detect,” Green said. “With these systems, which we call SNIPRs (for Single-Nucleotide-Specific Programmable Riboregulators), we have the capacity to identify any RNA sequence based on a single nucleotide difference.”

The technique is so sensitive, it can even detect epigenetic changes — subtle chemical modifications to genetic sequences that can regulate gene expression without changing the identity of individual bases.

“Advances in the method could one day be used as a low-cost alternative for personal genotyping,” according to Hao Yan, a coauthor of the new study and director of the Biodesign Center for Molecular Design and Biomimetics. “The simplicity of the technique may allow at-home screening for disease-linked mutations, providing rapid and accurate testing, while maintaining data privacy for users.”

In addition to its convenience as an inexpensive, versatile litmus test for mutation-related illness, the technique promises to shed new light on foundational issues in cell biology, including genetic resistance to antibiotics and mutations leading to the failure of frontline treatments for diseases like malaria and HIV.

The study appears in the current issue of the journal Cell.

portrait of man in lab

SNIPRs are RNA-based structures capable of identifying point mutations that can affect human health.

Alphabet of life

In humans, the genetic code is composed of some 3 billion pairs of nucleotides, arranged in the iconic DNA double helix. The language of nucleotides, which spell out the complete building plan for any living organism, including humans, is composed of sequences of just four letters, A, T, C and G, signifying the four bases of DNA.

Sequences of these four nucleotides form genes that provide the instructions for making proteins. Proteins provide structure to cells and tissues; including muscle, cartilage, ligaments, hair and skin. Proteins also supply the vital machinery of life, overseeing innumerable cellular processes, including metabolism, signal transmission, immune defense, food digestion and cell division. 

Hunting mutations

A point mutation in a DNA gene will be transcribed into RNA, sometimes disabling the resulting protein or altering its function, often with consequences for human health. To identify these mutations, the researchers designed SNIPRs — clever structures containing complementary snippets of RNA able to bind with RNA sequences in cells.

Within a cell, these structures activate when they encounter a mutated RNA sequence, dictated by the cell’s modified gene.

If the binding of a cell’s mutant RNA with the trigger strand is exact, the SNIPR unfolds, allowing sequence access by the ribosome — the machinery required to translate RNA into protein. If, however, the SNIPR encounters an unmutated sequence, there is a mismatch and translation of protein is blocked.

Proofreading RNA

The resulting 100-fold difference in gene expression between mutated and unmutated RNA sequences was observed in the bacterium E. coli, (as measured in protein production), making detection of point mutations easy.

The technique relies on keen detection of differences in so-called binding or hybridization energy.

“Typically, when you're thinking about a DNA or RNA base pairing, it's through hydrogen bonds,” Green said. “When G binds to C that's 3 hydrogen bonds and when A binds to U, that's two hydrogen bonds.”

In addition to point mutations, in vitro analysis can detect minor differences in binding energy when epigenetic changes like methylation occur.

The paper-based test can be used in the field in regions where medical resources are scarce. The technology holds particular promise for the developing world as it does not require elaborate equipment and can operate at human body temperature.

First-author Fan Hong, formerly with the Biodesign Institute and now a postdoctoral fellow at Harvard, designed computer algorithms that allow for the efficient design of SNIPRs based on desired RNA target sequences.

“To make SNIPRs easy to use, we automated the process so that everybody can design them without any knowledge of RNA folding and RNA interactions,” Hong said. “They already show lots of practical applications such as human genotyping, Zika virus detection and viral strain identification.”

RNA

SNIPRs are RNA-based structures capable of identifying point mutations that can affect human health.

Powerful technique a boon for science

Identifying particular strains is of vital importance epidemiologically. Some genetic variants of Zika for example, appear to pose greater risk of birth abnormalities, while the currently circulating coronavirus is also evolving and has a very similar sequence to the coronavirus that caused the SARS epidemic in 2002–03. Identifying the effects of these mutated pathogens and their geographic distribution is critically important in addressing these and future disease outbreaks.

The method could also offer new hope in the fight against cancer. For example, granulosa cell tumors, associated with a rare and aggressive form of ovarian cancer, result from just a single incorrect base out of the 3 billion nucleotide pairs that make up the genetic code, while the point mutations in tumor-suppressing BRCA1 and BRCA2 genes are responsible for a sixfold increase in the lifetime risk for breast cancer.

The fine-grained sensitivity of SNIPRs can discriminate between patients who are heterozygous or homozygous for given mutations, that is, whether they carry one or two copies of the mutated gene on their chromosomes, a critical factor in determining disease vulnerability.

Certain point mutations in HIV can lead to the failure of common antiretroviral therapies. A SNIPR test for such mutations could rapidly identify these mutations and guide appropriate treatment. Conventional tests for HIV drug resistance are prohibitively expensive for many in need, costing over $200 dollars per sample.

When SNIPR probes are combined with paper-based recognition systems, the potential for rapid, low-cost and precise detection of genetic point mutations can be extended globally, wherever such diagnostic tools are most critically needed. Additionally, SNIPRs promise to help researchers understand strain variations and mutation-linked resistance to common therapeutics.

In addition to their appointments with the Biodesign Institute, Green and Yan are both researchers at ASU’s School of Molecular Sciences.

Richard Harth

Science writer, Biodesign Institute at ASU

480-727-0378

 
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Giving back to the future

February 27, 2020

ASU students invest in outreach that helped them become engineers

When we ask kids what they want to be when they grow up, “engineer” isn’t usually among the top answers. Being an engineer isn’t as obvious as a doctor, athlete, teacher or other common dream careers. However, all these jobs rely on engineers, computer scientists and other technology professionals.

Introducing engineering concepts in fun ways and meeting engineering students and professionals can spark young people’s interest in these careers.

In fact, engaging outreach activities led many current engineering and computer science students to study at the Ira A. Fulton Schools of Engineering at Arizona State University. Some now return the favor and mentor young students through these same programs.

A pipeline for the next generation

The Fulton Schools conducts school-year and summer outreach activities for children as young as kindergarten. Students can learn how to code, make video games, build robots, explore sustainable energy production — all fun ways to introduce concepts they may never have associated with engineering. For even the youngest students, the experience means getting to practice being an engineer and planting the seed that this can be their future.

“At the elementary level and younger, outreach is about raising awareness for engineering. It’s about what engineering is and what engineers do,” said Jennifer Velez, coordinator senior of the Fulton Schools student outreach and recruitment programs. “It’s tapping into that curiosity and giving kids an opportunity to explore and develop what we call engineering habits of mind, which include creativity, collaboration and problem-solving.”

The youngest students accomplish this goal by tinkering and exploring, whereas older elementary and middle school students start to learn skills and apply them in coding and robotics projects.

Velez says it’s important to generate interest in engineering early, so by the time students are in middle and early high school they are on a technical academic path and have the foundational skills that will help them accelerate their engineering studies in college.

Many summer programs take place at ASU’s Tempe and Polytechnic campuses, where the young participants get to see university life and interact with undergraduate students. The experience makes engineering and the possibility of college in general feel more accessible.

“This exposure gives participants a comfort level and familiarity with the university — and it demystifies college a little bit,” Velez said. “That’s especially important for students who are underrepresented in STEM. They have this opportunity to ask questions, especially of their mentors and role models who look like them.”

Regardless of what the participants go on to do, they cultivate a new mindset and learn valuable skills that can be applied anywhere.

An early start to engineering

Along with running its own outreach activities, ASU facilitates robotics programs in Arizona for the nonprofit organization FIRST (For Inspiration and Recognition of Science and Technology).

From kindergarten through 12th grade, students can participate in multiple levels of robotics activities — FIRST Lego League Jr., FIRST Lego League, FIRST Tech Challenge and FIRST Robotics Competition. The organization uses these programs to encourage students to pursue engineering and STEM careers, and to become leaders and innovators with a strong grasp of work-life skills for the 21st century.

Arizona FIRST Lego League aims to “ignite an enthusiasm for discovery of the basic principles of science, technology, engineering, arts and math” by tasking teams of students aged 9 to 14 to solve community problems through robotics challenges. In the past year, more than 2,700 students across the state have participated in Arizona FIRST Lego League.

The Fulton Schools outreach program holds weeklong FIRST Lego League summer camps for beginner and intermediate levels of robotics knowledge. The university also helps with team activities that run from August through December, as well as helping to coordinate statewide tournaments for more than 360 teams throughout Arizona, including state championship tournaments held on ASU’s Tempe campus each January.

“For kids who have been intimidated by (robotics) or haven’t had the opportunity to try it, the summer camp takes away the mystery and it develops their excitement for these different projects,” said Laura Grosso, senior coordinator for Fulton Schools student outreach and retention programs. “From the kids’ perspective, it helps give them the experience and foundation to move on to a team.”

Zach Smith, a first-year computer science student at ASU, says he knew he wanted a computer-related career as early as third grade. That’s when he began participating in FIRST Lego League in Flagstaff, Arizona, and realized not only could he become an engineer, but that elementary school wasn’t too early to start building his skillset.

Smith notes that learning to program a Lego robot to navigate through challenges helped him to pinpoint which area of computer science most interested him.

Grosso said, “People who have been through this program learn skills that are critical for engineers — not just the hard skills and the ability to understand complicated technical concepts, but the courage to tackle these really challenging areas of study.”

As Smith moved through the FIRST ranks and participated in the more advanced FIRST Robotics Competition during high school, he also strengthened his technical and teamwork skills.

He also became familiar with the college engineering experience as his team competed in tournaments hosted at ASU, where current Fulton Schools undergraduate students gave firsthand accounts about their experiences. Seeing what life could be like as a computer science student at ASU made him all the more eager to become a Sun Devil.

Seeing how engineering impacts society

While some students have an early interest in engineering, others get a later start.

The Fulton Schools outreach team aims to help middle school and high school students see themselves as engineers through a beginner version of one of its college extracurriculars, the Engineering Projects in Community Service, or EPICS, program.

EPICS enables college students to solve real-world community challenges through service learning projects. The same concept is used by 10 middle schools and 19 high schools in EPICS High.

Some schools use the program as a way to introduce engineering to middle school and first-year high school students, while others use it to help advanced junior and senior students apply technical skills they’ve already learned over the course of their STEM education, or anywhere in between. More than 700 students currently participate in EPICS High in Arizona.

Typically, students work with their teachers and not-for-profit organizations to make an impact in their community with engineering concepts and human-centered design skills.

“Students can deliver a complete project, and it is important at that age for them to experience failure, learn to push through failures and iterate to deliver something,” Velez said. “It allows them to feel good about their success, which is a strong motivation to continue into engineering.”

Seeing how engineering tackles issues that students see in their own communities also helps them create an engineering identity and see themselves in engineering careers down the line.

Some EPICS High groups also have ASU student mentors helping to guide the middle- and high-schoolers to learn skills they need to create successful community solutions.

Seth Mazza, a second-year aerospace engineering major in the Fulton Schools, was already interested in engineering, but his two years of EPICS High experience at MET Professional Academy in Peoria, Arizona, gave him a new perspective and motivation to pursue it as a career.

He worked on two projects: a modular electric piano keyboard and an elastic wristband to help people with hand and arm impairments more easily use wearable devices.

“One of the most memorable moments for both of these projects was reaching out to potential stakeholders and getting real validation of the idea,” Mazza said. “These experiences also made me want to attend ASU even more and join the EPICS program at the college level.”

Creating a culture of giving back

Not only did their experiences help lead Smith and Mazza to study computer science and aerospace engineering at ASU, they also encouraged them to give back and return to the same outreach programs as mentors.

“Students who had an EPICS High mentor seem more likely to become mentors themselves,” Velez said. “They value those relationships.”

Velez says when students come back to mentor in the same program, it shows how influential and valuable the experiences were when the mentors were participants.

“It shows how involved and invested they were in their projects when they were in EPICS High, and now they’re excited to help other students get the same kind of experience.”

Mazza, who has now been an EPICS High mentor for two years at the same high school he attended, exemplifies this idea.

“I wanted to make sure other EPICS High students gained everything I did in the program and more,” he said. “I can help them through the rough patches of a project without taking away the learning experiences.” 

Mentors also feel like they’re making a difference in the lives of others, and for themselves. For Mazza, mentorship is often an inspiring experience.

“Any day where I can get students to come to some sort of realization and figure out what they were stuck on or missing is a great day,” Mazza said. “The way their faces light up when they reach said realization alone, is enough to make me want to continue being a mentor.”

For others, helping people is the core value they are most drawn to. FIRST programs emphasize the importance of support just as much as technical skills.

FIRST mentors — who range from high school and college students to industry professionals — play a key role in imparting the value of teamwork. Smith’s FIRST mentor was a high school student who ended up earning a master’s degree in software engineering from ASU. Smith remembers his mentor’s enthusiasm influenced his desire to “pay it forward in the future.” 

Now, as a mentor himself, Smith works with his FLL team of elementary school students who are the same age he was when he started participating. He teaches them important lessons for all areas of robotics competition: robots, research projects and core values.

Like the college students he met at youth competitions hosted by ASU, Smith also enjoys talking with teams and “showing the kids that college is something they can attain in the future.”

Overall, it was a valuable experience when he was in third grade, and it continues to be rewarding as a mentor.

“Seeing these students grow in the same way I did gives me hope for future engineers,” Smith said. “I know they have the skills necessary to solve the problems of tomorrow.”

Top photo: Elementary and middle school students participate in the FIRST LEgo League state championship tournament held at Arizona State University. Outreach programs such as FIRST Lego League and Engineering Projects in Community Service for middle and high school students, known as EPICS High, help introduce complex engineering concepts in a fun way starting at an early age. Photo by Connor McKee/ASU

Monique Clement

Communications specialist , Ira A. Fulton Schools of Engineering

480-727-1958