In the bustling world of cellular machinery, a revolutionary high-throughput assay is turning bacteria into tiny factories for designing next-generation nanotechnology.
Imagine being able to design a microscopic gateway so precise that it could identify individual molecules as they pass through—a biological version of a barcode scanner. This is the promise of protein nanopores, nanoscale channels that control the flow of substances across cell membranes. For years, studying and engineering these remarkable structures has been a slow, laborious process, hampered by a fundamental technological gap: the inability to combine detailed functional analysis with high-throughput experimentation. Addressing this challenge, scientists have developed a powerful new method called the functional nanopore (FuN) screen, a versatile assay that is accelerating our ability to study and engineer protein nanopores within the common bacterium Escherichia coli 1 6 .
Nanopores are nature's sophisticated gatekeepers. These protein-based channels are embedded in cell membranes and perform a range of vital physiological functions, from signaling to transport 1 . Beyond their biological roles, nanopores have emerged as transformative tools in biotechnology. Most famously, they form the basis of nanopore sequencing technology, which allows for the direct, real-time reading of DNA and RNA strands 4 5 .
Direct, real-time reading of genetic material
A nanoscale pore is set in a membrane separating two fluid chambers. When a voltage is applied, ions flow through the pore, creating a measurable current.
As a molecule like DNA or a protein passes through (a process called translocation), it disrupts this current in a characteristic way.
The disruption produces a unique electrical signature—a current blockade—that can be decoded to identify the molecule 4 .
Scientists are now pushing this technology toward an even grander challenge: protein sequencing. While sequencing DNA has become routine, determining the precise order of amino acids in a protein remains remarkably difficult. Existing methods like mass spectrometry have limitations in sensitivity and the ability to detect rare modifications 4 . Single-molecule protein sequencing using nanopores could revolutionize our understanding of cellular machinery, precision diagnostics, and the development of new therapies 4 5 . However, achieving this requires the ability to design nanopores with exact specifications—a capability the FuN screen now provides.
Before the FuN screen, the study of protein nanopores was caught in a methodological bind. Techniques like electrophysiology could provide high-resolution, single-molecule data on nanopore function but were notoriously low-throughput, allowing researchers to study only a few variants at a time 1 6 .
This presented a major obstacle for nanopore engineering, which often relies on creating and testing thousands of mutant versions to find ones with improved properties.
This gap hindered progress in both basic science and applied biotechnology. Without a capacity for combinatorial screening, researchers lacked an efficient way to explore the vast "sequence space" of possible nanopore variants or to perform detailed studies of the molecular features governing nanopore assembly and stability 1 . The FuN screen, first described in detail in 2022, was developed specifically to bridge this gap, offering a quantitative and dynamic readout of nanopore function with the ease, scalability, and throughput of a cellular assay 1 6 .
The FuN screen is a clever genetic assay that turns E. coli into a living nanopore testbed. Its core innovation lies in using genetically encoded fluorescent protein sensors to detect the functional activity of nanopores in the inner membrane of the bacterium 1 .
| Step | Key Component | Function in the Assay |
|---|---|---|
| 1 | E. coli Inner Membrane | Serves as the natural environment for nanopore assembly and function. |
| 2 | Engineered Nanopore Gene | Provides the genetic blueprint for the nanopore protein to be tested. |
| 3 | Calcium Ions (Ca²⁺) | Act as the measurable ion that flows through functional nanopores. |
| 4 | Fluorescent Sensor | Detects the rise in intracellular calcium and converts it to a fluorescent signal. |
| 5 | High-Throughput Analyzer | Measures fluorescence across thousands of cells, quantifying nanopore activity. |
Researchers introduce genes encoding the nanopore proteins to be studied into E. coli cells. These nanopores are expressed and assemble in the bacterium's inner membrane.
The assay capitalizes on the fact that the E. coli cytoplasm is naturally low in calcium. When a functional nanopore forms, it creates a channel that allows calcium ions (Ca²⁺) from the external environment to flow into the cell.
Inside the cell, a special fluorescent sensor protein waits. When calcium levels rise due to nanopore activity, the sensor binds to calcium and undergoes a change, emitting a fluorescent signal.
The intensity of this fluorescence, which can be measured automatically using high-throughput systems like flow cytometers or plate readers, provides a direct, quantitative measure of nanopore assembly and function. A brighter signal indicates more calcium influx and, therefore, a more functional nanopore 1 .
This process transforms the abstract question of "Is this nanopore working?" into a simple, measurable output—fluorescence—that can be used to rapidly screen thousands of different nanopore variants simultaneously.
To illustrate the power of the FuN screen, researchers conducted a sophisticated protein engineering experiment. They set out to investigate the structural plasticity of nanopores—specifically, to see if they could engineer functional nanopores with different numbers of subunits, thereby controlling the size and geometry of the pore 1 .
| Engineering Aspect | Question Investigated | Key Finding from FuN Screen |
|---|---|---|
| Structural Plasticity | Can a transmembrane module form pores with different subunit numbers? | Yes, the S2168 module successfully formed hexamers, heptamers, and octamers. |
| Linker Role | How do connecting sequences impact the final nanopore? | Linkers are crucial for fine-tuning the functional properties of the engineered pore. |
| Assay Versatility | Can the screen handle complex protein engineering? | Yes, it efficiently identified functional combinations from a large, diverse library. |
Advancing the field of nanopore biotechnology relies on a suite of specialized tools and reagents. The following table outlines some of the key components used in techniques like the FuN screen and related nanopore research.
| Tool or Reagent | Function in Research | Example Use in FuN Screen |
|---|---|---|
| Fluorescent Biosensors | Genetically encoded proteins that detect specific ions or molecules. | Detects Ca²⁺ influx as a proxy for nanopore activity 1 . |
| Oligomerization Domains | Protein modules with defined geometric structures (e.g., hexameric). | Provides the structural blueprint for engineered pore geometry 1 . |
| Linker Peptides | Short, flexible amino acid sequences connecting protein domains. | Fine-tunes function in engineered chimeric nanopores 1 6 . |
| Cellular Expression Systems | Living cells (like E. coli) used to produce and host proteins. | Provides the membrane environment for nanopore assembly and testing 1 . |
| High-Throughput Screens | Assays that quantitatively analyze thousands of variants in parallel. | Enables rapid functional analysis of entire nanopore libraries 1 . |
The development of the FuN screen represents a significant leap forward, but it is part of a broader trend in biotechnology that combines ultrahigh-throughput experimentation with advanced data analysis. The miniaturization of assays into picoliter-volume droplets, for instance, allows researchers to screen millions of protein variants in a single day, generating massive sequence-function datasets .
These vast datasets are increasingly being interpreted with the help of machine learning (ML) algorithms. The goal is to construct detailed "fitness landscapes" that predict how specific changes to a protein's sequence will affect its function .
This powerful synergy between experimentation and computation promises to accelerate the design of nanopores for a wide range of applications.
Designing nanopores that can detect specific chemical threats, disease biomarkers, or environmental pollutants with high sensitivity 5 .
Engineering nanopores that can be incorporated into synthetic cells or vesicles for controlled release of therapeutic agents.
As these tools continue to evolve, the vision of designing custom nanopores on demand for specific tasks in biomedicine, synthetic biology, and materials science is rapidly moving from the realm of science fiction to tangible reality. The FuN screen has provided a critical key, unlocking a high-throughput path to explore and engineer nature's most sophisticated gatekeepers.