How Scientists Are Turning Nanopores into Peptide Sequencing Devices
By combining bacterial nanopores with molecular motors, researchers are unlocking the ability to read proteins at single-molecule resolution
Imagine trying to read a secret code by pulling a string of letters through a tiny hole too quickly to see any of them. This is essentially the challenge scientists have faced in their quest to sequence proteins, the complex molecules that perform nearly every function in our bodies. While DNA sequencing has been revolutionized by technologies that can read genetic code rapidly and inexpensively, protein sequencing has lagged significantly behind, remaining complex, costly, and limited in capability. But what if we could thread a single protein molecule through a nanoscale pore and read its composition like a scanner deciphering a barcode?
Proteins have varied charges and complex 3D structures that make them difficult to sequence compared to the uniform DNA backbone.
Combining bacterial nanopores with molecular motors to control peptide movement for accurate amino acid reading.
In a groundbreaking study published in Chemical Science, researchers have done just that. By combining a specially engineered nanopore from bacteria with a molecular motor protein, they've developed a system that can control the movement of peptides (short protein fragments) and distinguish between individual amino acid building blocks. This breakthrough approach could eventually enable us to sequence entire proteins in their natural form, opening new frontiers in understanding diseases, developing drugs, and unraveling the fundamental mechanics of life itself 1 .
From DNA Reading to Protein Sensing
To appreciate this achievement, we first need to understand how nanopore sensing works. Picture a extremely tiny hole—just wide enough for a single molecule to pass through—created in a membrane that separates two chambers of salt water. When we apply an electric voltage across this membrane, ions flow through the pore, creating a measurable electrical current. As molecules pass through this nanoscale opening, they partially block the ion flow, causing characteristic dips in the current. Crucially, different molecules create distinct electrical signatures based on their size, shape, and electrical properties 5 6 .
Molecules passing through the nanopore create unique electrical signatures
This ingenious principle has already transformed DNA sequencing. Commercial nanopore devices can now sequence entire genomes by measuring how each of the four DNA nucleotides (A, C, G, T) uniquely modulates the ionic current as they pass through a pore 3 . However, applying this same approach to proteins has proven far more challenging for several reasons:
Unlike the uniformly charged DNA backbone that responds predictably to electric fields, proteins have varied charges and complex three-dimensional structures that often need to be unfolded before sequencing 5 .
Proteins are built from 20 different amino acids (compared to just 4 DNA bases), making discrimination more difficult.
Perfect Dimensions for Molecular Sensing
The recent breakthrough hinges on a special bacterial pore called Mycobacterium smegmatis porin A (MspA). This nanopore possesses an almost ideal geometry for molecular sensing. While other protein pores like alpha-hemolysin have long, narrow channels that blur the signal from multiple nucleotides at once, MspA features a short, sharp constriction zone—just 0.6 nanometers long and 1.2 nanometers wide—that's perfectly sized to read single nucleotides or amino acids 6 .
Think of the difference between trying to read a sign while driving past at high speed versus while stopped at a traffic light. MspA's structure gives us that crucial "stopped" moment by focusing the electrical field on just a few amino acids at a time.
Goblet-shaped structure with constriction zone
Additionally, MspA is incredibly stable—it can withstand extreme temperatures, pH levels, and denaturing agents that would destroy most biological molecules 8 .
Through genetic engineering, scientists created a mutant version called M1-NNN-MspA by replacing three aspartic acids with neutral asparagines. This modified pore readily allows single-stranded DNA and peptides to pass through while maintaining its exquisite sensitivity to molecular features 3 6 .
Nature's Molecular Control Mechanism
Even with an ideal pore, molecules still pass through too quickly to be read accurately. The solution? Borrow a molecular motor from nature's toolkit to ratchet the peptide through the pore in controlled, stepwise motions.
Enter helicases—remarkable enzymes that normally unwind double-stranded DNA into single strands during cellular processes like DNA replication and repair. These are true molecular machines that convert chemical energy from ATP (the cellular energy currency) into mechanical movement along DNA strands. Helicases can be thought of as nucleic acid motors that translocate along single-stranded nucleic acids and, upon reaching a junction, continue to move through and separate the duplex 2 .
Certain helicases, like those from bacteriophage T7, are particularly impressive—they can translocate along DNA at rates of 132 bases per second while being incredibly processive, traveling an average of 75 kilobases before dissociating. This means they can maintain their grip on the DNA strand over remarkably long distances 4 .
Researchers realized that by hijacking this natural motor function, they could use helicases to control the speed at which peptide-conjugated DNA moves through the MspA nanopore. The helicase acts like a precise winch, pulling the molecule through the pore at a manageable pace that allows each amino acid to be "read" 1 .
Peptide Sequencing with MspA and Helicase
In their landmark 2021 study, Chen and colleagues devised an elegant system that brings all these components together. Their goal was to demonstrate that a helicase motor could control peptide movement through MspA well enough to distinguish between different amino acids—a crucial step toward true protein sequencing 1 .
The researchers chemically attached the target peptides (up to 17 amino acids long) to a handle made of single-stranded DNA. This clever conjugation creates a hybrid molecule that helicases can grip and move along, while the peptide portion produces detectable signals in the nanopore.
A helicase motor was loaded onto the ssDNA portion of the conjugate. In the presence of ATP, the helicase begins moving along the DNA strand in its natural direction.
The MspA nanopore was embedded in a lipid membrane separating two chambers. When voltage is applied, the charged DNA-peptide conjugate is drawn toward the pore.
As the helicase motor moves along the DNA, it pushes or pulls the conjugated peptide through the MspA constriction zone in a controlled, stepwise manner—far slower than the uncontrolled translocation that would occur with voltage alone.
As each amino acid passes through the pore's constriction, it creates a characteristic disruption in the ionic current. These signals are recorded and analyzed to identify specific amino acid features 1 .
The results were striking. The system successfully distinguished between amino acid residues of different charges—a crucial capability since charge is a fundamental property that influences protein function. Even more impressively, it could differentiate between identical amino acids that had undergone different post-translational modifications, specifically identifying varying phosphorylation sites 1 .
Phosphorylation (the addition of phosphate groups to proteins) is one of the most important regulatory modifications in biology, controlling when proteins are active or inactive. The ability to detect it at the single-molecule level represents a significant advance. The researchers achieved a read length of up to 17 amino acids, demonstrating the potential for reading meaningful protein segments rather than just short fragments 1 .
| Aspect | Achievement | Significance |
|---|---|---|
| Read Length | Up to 17 amino acids | Demonstrates capacity for meaningful protein segments, not just tiny fragments |
| Discrimination Ability | Amino acids of different charges | Charge is a fundamental property influencing protein structure and function |
| Modification Detection | Distinct phosphorylation sites | Enables identification of crucial regulatory protein modifications |
| Control Mechanism | Helicase-controlled translocation | Provides solution to the critical challenge of uncontrolled molecule movement |
Essential Research Reagents and Materials
Bringing this cutting-edge technology to life requires specialized biological and chemical components. Each element plays a crucial role in the sophisticated molecular dance of nanopore sequencing.
| Research Tool | Function and Importance |
|---|---|
| MspA Nanopore (M1-NNN mutant) | Engineered protein pore with ideal geometry for single-amino acid sensitivity; modified constriction zone allows peptide translocation 3 6 |
| Helicase Motor (e.g., T7 helicase) | Molecular motor that uses ATP hydrolysis to control peptide-DNA conjugate movement through the pore; enables stepwise translocation 1 4 |
| ssDNA-Peptide Conjugates | Hybrid molecules that allow helicases to grip and move peptides through the nanopore; crucial for coupling motor function to peptide sequencing 1 |
| ATP and Buffer System | Provides chemical energy for helicase movement and maintains proper ionic conditions for nanopore current measurements 2 |
| Lipid Bilayer System | Creates the artificial membrane that houses the nanopore; typically uses diphytanoyl phosphatidylcholine (DPhPC) for stability 7 |
| Electrical Recording Setup | High-sensitivity equipment to measure picoampere-scale current changes as molecules pass through the nanopore 5 6 |
Implications and Future Directions
While the controlled movement of ssDNA-conjugated peptides through MspA represents a spectacular proof of concept, researchers acknowledge that several challenges remain before this technology can sequence entire proteins. The resolution needs further improvement to distinguish all 20 amino acids reliably, not just those with different charges or modifications. The researchers suggest that this may require further engineering of MspA's constriction zone to better stretch the target peptide and minimize random thermal motion 1 .
The potential applications of this technology are vast. A robust protein sequencing method could revolutionize personalized medicine by allowing comprehensive analysis of an individual's complete protein profile (proteome). It could detect protein biomarkers for early disease diagnosis, identify novel drug targets, and help monitor therapeutic responses. In basic research, it could uncover the vast landscape of proteoforms—different molecular versions of proteins created through genetic variation, alternative splicing, and post-translational modifications 5 .
The impact of this research extends beyond protein sequencing alone. The combination of MspA with molecular motors has created what scientists call Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), a powerful tool for studying enzyme mechanisms at unprecedented resolution. SPRNT can detect enzymatic motions along DNA with 40-picometer precision on sub-millisecond timescales, allowing researchers to observe how molecular motors interact with their substrates in real time 6 .
Recent advances suggest we're on the cusp of even more dramatic breakthroughs. A 2024 study in Nature demonstrated a method for reading long protein strands multiple times using a different biological nanopore (CsgG) and the ClpX unfoldase motor. This multi-pass approach significantly improves sequencing accuracy and has successfully identified single-amino-acid substitutions and post-translational modifications in strands hundreds of amino acids long .
The controlled movement of peptides through MspA nanopores using helicase motors represents more than just a technical achievement—it's a glimpse into a future where we can read the language of proteins as easily as we now read genetic code. Just as the Human Genome Project transformed our understanding of biology and medicine, the ability to sequence entire proteomes could unlock secrets of cellular function that have remained hidden until now.
From distinguishing phosphorylated amino acids to unfolding the complex structures of functional protein domains, this technology continues to evolve at a remarkable pace. While challenges remain, the foundation is firmly established. The molecular scanners that once existed only in theory are now taking shape in laboratories worldwide, promising a new era of scientific discovery powered by our ability to read the intricate molecular machinery of life—one amino acid at a time.