The first draft of the human genome took more than a decade of research and cost some $3 billion. Today, new techniques for high-speed DNA sequencing have radically reduced the time and cost to decrypt roughly 3 billion base pairs that form the blueprint for human life.
Yet the human genome is far from the whole story. To fully understand the biological underpinnings of health and disease, scientists must probe the world of proteins — large, complex molecules required for the structure, function and regulation of the body's tissues and organs.
Now, Chao Wang, a researcher with the Biodesign Center for Molecular Design and Biomimetics at Arizona State University, and his colleagues are advancing an ambitious project: developing new methods for sequencing individual protein molecules using a rapid, accurate and inexpensive method.
Single-molecule protein sequencing of this kind holds the potential to revolutionize diagnostic medicine through the identification of protein biomarkers for cancer and other deadly diseases, provide earlier and more accurate diagnoses and deepen our understanding of how healthy cells function.
“Sequencing proteins and analyzing their post-translational modifications are particularly important for the studies of heart disease, cancer, neurodegenerative diseases and diabetes,” says Wang, who is also an associate professor in the School of Electrical, Computer and Energy Engineering at ASU. “One important future use of this technology is single-cell level proteomic analysis, which can have profound impact on both disease diagnostics and therapeutics.”
Multipurpose building blocks
In addition to maintaining proper biological functioning of cells and tissues throughout the body, protein interactions are implicated in many diseases. Ideally, researchers would like to read protein sequences with the ease of current DNA sequencing methods, investigating the complexities of protein behavior and finding new therapies for a broad range of protein-linked maladies, including cystic fibrosis, diabetes, cancer, Alzheimer's disease and Parkinson's disease.
Until now however, the goal has proved challenging and elusive.
Unlike DNA, which is composed of just four nucleotide letters, proteins are made up of some 20 different amino acids, which bind together to form sequences, before folding into complex 3D configurations. These 20 amino acids arrange themselves to form tens of thousands of proteins in the body.
To further this research, Wang has received the prestigious NIH Director’s New Innovator Award. In 2022, the NIH awarded over $200 million to support transformative biomedical research projects under four research categories. The NIH Director's New Innovator Award was established in 2007 as part of its High-Risk, High-Reward Research program to “accelerate the pace of biomedical, behavioral and social science discoveries by supporting exceptionally creative scientists with highly innovative research.”
“This award is indeed a great honor and surprise. The opportunities ASU provided to me during the past few years have really allowed me to work across disciplinary boundaries to pursue crazy but scientifically meaningful ideas,” Wang says. “This award certainly marks a new beginning for my journey ahead. It provides not only the funds that are otherwise difficult to obtain for this project, but also more confidence to continue my multidisciplinary path in both research and education.”
Stephen Phillips, director of the School of Electrical, Computer and Energy Engineering, notes that electrical engineers don’t often earn this award.
“Chao Wang’s challenging work across several disciplines in engineering, biology and health reflects the impressive level of innovation and creativity that this award is intended to further,” Phillips says. “His ongoing efforts to leverage transdisciplinary ideas to accomplish new results is sure to inspire others to follow his lead.”
Sequence of innovations
Currently, the gold standard for sequencing proteins is mass spectrometry, but the method has a variety of limitations. For many applications, mass spectrometry is simply not sensitive enough. The technique, which requires bulky, expensive machinery and technological expertise, requires about a million protein copies in a sample to make an accurate detection.
Researchers would like to identify individual protein molecules, which would allow the ferreting out of details particular to each separate protein, including modifications that may affect the protein’s function.
To make single-molecule protein sequencing an affordable and practical tool, several difficulties plaguing current efforts must be overcome. These include sensing system reliability, slow readout, high noise and high cost.
The new project addresses these issues with the design of an on-chip integrated, electronic system. The proteins are read sequentially using a low noise nanopore composed of sapphire for greater accuracy.
The device is outfitted with additional photonic nanostructures, electronic device components, and circuit elements capable of translating single-molecule protein fingerprints into electronic signals. This synchronous recording will improve the accuracy, potentially enabling the identification of all 20 amino acids at a higher throughput than existing methods.
The researchers will further improve protein identification using deep-learning algorithms. The result is a portable device capable of rapid, accurate, inexpensive protein sequencing, poised to improve data throughput by two to three orders of magnitude.
The idea of using nanopores for sequencing has been around awhile, and several variants of the method have been used successfully for DNA sequencing. The technique feeds sequences of DNA nucleotides or, in the case of proteins, amino acids, through a very narrow pore, much like a length of thread passing through a needle’s eyelet. The nanopore must be fabricated a few billionths of a nanometer in diameter. A detector then scans each member of the DNA or protein sequence as it passes through the nanopore.
The new single-molecule protein sequencing technique makes use of a very low-noise, all-sapphire nanopore fluidic device, as well as two different sensing modes, which combined dramatically improve the accuracy of the amino acid identifications. The use of sapphire also provides improved structural stability over other materials, a crucial consideration for a device that must be engineered to exacting standards at nanoscale dimensions.
Although the amino acids in the protein sequence are fluorescently tagged, the method does not require expensive and labor-intensive fluorescent microscopy to read the signals, which are picked up by the optoelectronic channel of the circuit.
The use of advanced semiconductor chip design and manufacturing technologies has the potential to lower the costs, decrease the instrument size and directly digitize data for biology and health care use. Such advances can accelerate translation between bench and bedside.
“Chao’s research represents a deeply creative marriage of innovative nanotechnology with fresh insights in the life sciences,” said Hao Yan, director of the Biodesign Center for Molecular Design and Biomimetics, noting that Wang is now the fourth recipient from his center to land this prestigious NIH Director’s New Innovator award.
“I am thrilled at the announcement of this much-deserved award and believe this research holds the potential for transformative advances in medicine, particularly for the early and accurate detection of disease,” Yan said.
The single-molecule protein sequencing device is Wang’s latest nanobiotechnology creation. In earlier research, he demonstrated the power of combining semiconductor and nanophotonic technologies to design faster, more accurate, less expensive and more broadly accessible biosensing technologies, including portable devices for the detection of infectious agents, such as Ebola and SARS CoV-2, as well as for African swine fever.
Grant ID: DP2-GM149552
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