In the world of the extremely small, scientists have devised a molecular fishing rod to catch and read the building blocks of life.
Imagine being able to detect a single molecule—one of the fundamental units of life—and identify its specific structure in real-time.
This is not science fiction but the reality of nanopore tweezing, a cutting-edge technique that is pushing the boundaries of single-molecule science. By combining the precision of nanopores with the unique properties of peptide nucleic acids (PNAs), researchers are developing powerful new tools for biological discovery, with the potential to revolutionize how we diagnose diseases and understand genetics at the most fundamental level 1 3 .
To appreciate the innovation of nanopore tweezing, it's helpful to understand its key components.
A synthetic molecule that mimics DNA. Like DNA, it can carry the genetic codes of nucleobases (A, T, C, G), but its backbone is completely different. Instead of the sugar-phosphate backbone of DNA, PNA has a structurally similar but uncharged pseudopeptide backbone 1 3 .
This lack of charge is crucial. It makes PNAs less "slippery" than DNA in certain electrical environments, allowing them to be held in place more easily for detailed inspection. Since their discovery, PNAs have shown tremendous potential in antigene and antisense therapy, functional genomics, and as probes for diagnosis and detection 1 .
A tiny hole, typically just nanometers in diameter. In sequencing technologies, a nanopore is set within a membrane that separates two chambers filled with an ionic solution. When a voltage is applied across the membrane, ions flow through the pore, creating a measurable electrical current 1 3 9 .
The revolutionary concept, first demonstrated in the 1990s using a protein nanopore from Staphylococcus aureus called α-hemolysin (α-HL), is that when a single DNA or PNA molecule is driven through the pore, it disrupts the current in a unique, measurable way 1 3 9 .
The challenge has always been speed. Molecules like ssDNA can shoot through a nanopore at rates of about one nucleotide per microsecond, making it difficult to get a clear "read" of each base 1 . This is where the "tweezing" part comes in.
The core idea behind the nanopore-tweezer approach is ingenious yet simple: if you can't read the molecule as it zips through, find a way to hold it steady.
Researchers engineered PNA molecules with a special structure, attaching segments of positively charged lysine amino acids to one end and segments of negatively charged glutamic acid to the other end 1 3 .
This created a synthetic molecular "dipole" with distinct positive and negative ends 1 3 .
When this charged PNA is captured by the α-HL nanopore and a voltage is applied, an electrostatic tug-of-war ensues. The positive charges are pulled toward the negative electrode, while the negative charges are pulled toward the positive electrode 1 .
The opposing forces trap the PNA inside the pore, significantly increasing its residence time and allowing for a prolonged, detailed examination 1 . This is the nanopore tweezer in action.
Electrostatic Forces
Opposing electrostatic forces hold the PNA molecule in place within the nanopore.
In a pivotal 2021 study, researchers set out to demonstrate that this tweezing system could not only capture PNA but also distinguish between its individual nucleobases 1 3 .
The team designed a series of specialized PNA constructs, each with a middle section composed of a specific sequence of nucleobases, flanked by the charged lysine and glutamic acid segments.
Preparing the Stage
A lipid bilayer membrane was formed, creating a barrier between two electrolyte-filled chambers. A single α-hemolysin protein nanopore was inserted into this membrane 1 .
Introducing the Actor
The engineered PNA constructs were added to the specific side of the membrane (trans side) that would facilitate their optimal capture, guided by attractive electrostatic interactions 1 .
Running the Show
A transmembrane voltage was applied, driving the ionic current and initiating the capture of the PNA molecules. As each PNA was caught in the nanopore's tweezing action, the fluctuations in the ionic current were recorded with high precision 1 .
Decoding the Signal
The unique current blockade signatures generated by each type of PNA were analyzed. By studying these patterns, researchers could infer the composition and order of the nucleobases in the PNA's structure 1 .
The following table details the key materials used in this experiment and their functions 1 :
| Research Reagent | Function in the Experiment |
|---|---|
| α-hemolysin (α-HL) protein | Forms the biological nanopore embedded in the lipid membrane; the core sensing element. |
| Engineered PNAs | The molecules under investigation; their nucleobase sequence is the target for detection. |
| DPhPC Lipid | Used to form the artificial bilayer membrane that houses the nanopore. |
| KCl Electrolyte Solution | Creates the ionic environment necessary for conducting an electrical current through the nanopore. |
| HEPES/Tris Buffers | Maintains a stable pH level in the solution, crucial for consistent protein and PNA behavior. |
The table below lists the specific PNA constructs designed for this study, illustrating the variety of sequences tested 1 :
| PNA Construct | Nucleobase Sequence (5' to 3') |
|---|---|
| PN1 | GGG-GGG |
| PN2 | TTT-TTT-TTT-TTT |
| PN3 | AAA-AAA-AAA-AAA |
| PN4 | CCC-CCC-CCC-CCC |
| PN5 | GGG-TTT-GGG-TTT |
| PN6 | TTT-GGG-TTT-GGG |
The experiment was a success. The system demonstrated its ability to detect distinct bases on homopolymeric PNA (strands with a single type of base) and could identify triplet bases on more complex, heteropolymeric strands 1 . The different nucleobases, due to their unique physical shapes and chemical properties, caused characteristically different disruptions in the ionic current as they occupied the narrow constriction of the nanopore.
Simulated representation of how different PNA sequences create unique current blockade signatures when trapped in a nanopore.
The analysis of the current blockades revealed rich insights into the conformational dynamics of the PNA within the nanopore. This deeper understanding is vital for perfecting the recognition capability needed for true single-molecule sequencing 1 . The key findings and their importance are summarized in the table below.
| Key Finding | Scientific Importance |
|---|---|
| Detection of distinct bases on homopolymeric PNA | Confirms the technique's fundamental sensitivity to base identity. |
| Identification of triplet bases on heteropolymeric strands | Demonstrates the potential for resolving short sequences, a step towards full sequencing. |
| Observation of PNA conformational dynamics | Provides crucial information for refining models and improving measurement accuracy. |
The nanopore tweezing-based detection of nucleobases on PNA is more than a laboratory curiosity; it is a significant "proof-of-concept" that enriches the growing toolkit of single-molecule analysis 1 . While commercial nanopore sequencing technologies from companies like Oxford Nanopore Technologies have already made massive leaps in reading native DNA and RNA by other means, the fundamental research into techniques like tweezing provides deep insights that fuel further innovation 4 5 .
Ultra-sensitive devices that can detect specific genetic markers for diseases.
Focused analysis of particular genes of interest without the need to sequence an entire genome.
The same principles are now being explored to tackle the even more complex challenge of sequencing proteins, a feat that could dramatically advance our understanding of cellular machinery and disease mechanisms 4 .
As these technologies continue to mature, moving from academic labs to real-world applications, the ability to "tweeze" and read the molecules of life one by one will undoubtedly unlock new frontiers in medicine, biology, and beyond.