Biological Nanopores: Engineering on Demand

Abstract

Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions.

Keywords: aptamers; nanopores; oligomerization; pore-forming toxins; sensing.

Figure 1

An illustration of a biological nanopore and the principle of nanopore-based biosensing. Direct sensing relies on the distinct current signatures produced by the translocation of individual analytes (top right). Indirect sensing employs an additional, adapter molecule that specifically recognizes the analyte (bottom right). The adapter (shown in red) specifically interacts with the analyte (yellow) and the translocation of the adapter•analyte complex results in a unique current signal.

Figure 2

Cross-sections and overall dimensions of the most frequently used protein nanopores. “Ex” and “in” denote the extracellular and intracellular sides, respectively. Individual monomers are highlighted in yellow. Dimension are given for the total length of the pore and the narrowest part of the transmembrane channel (i.e., the sensing region). Outer membrane protein F (OmpF): a single β-barrel of the E. coli outer membrane protein F (PDB ID 2ZFG), OmpG: E. coli outer membrane protein G (PDB ID 2JQY), CsgG: E. coli curli transport channel CsgG (PDB ID 4UV3), MspA: M. smegmatis porin A (PDB ID 1UUN), α-HL: S. aureus α-hemolysin (PDB ID 7AHL), FhuA: E. coli ferric hydroxamate uptake component A (PDB ID 1BY3), AeL: A. hydrophila aerolysin (PDB ID 5JZH), FraC: A. fragacea fragaceatoxin C (PDB ID 4TSY), Lys: Eisenia fetida lysenin (PDB ID 5EC5), φ29p: φ29 phage DNA packaging motor (PDB ID 1FOU), ClyA: S. enterica cytolysin A (PDB ID 6MRU), PlyAB: Pleurotus ostreatus pleurotolysin (PDB ID 4V2T).

Figure 3

A generalized laboratory pipeline for the production of protein pores from pore forming toxins. Following expression in E. coli, the soluble monomers (shown in yellow) are isolated via chromatography. Addition of small or large unilamellar vesicles (SUV or LUV, respectively) induces spontaneous oligomerization and pore formation (a top view of a theoretical octamer is shown). Upon disruption of liposomes by addition of detergent, the mature pores are purified to remove unreacted monomers and improperly assembled pores.

Figure 4

(A) A schematic representation of a multiple-level blockade. The smaller amplitude is highlighted in purple, the larger in gold. (B) Loss of information due to over-filtering.

Figure 5

Controlling the speed of DNA translocation with a φ29 DNA polymerase (DNAP). When analyzed alone, the DNA to be sequenced (in black) translocates too rapidly for individual bases to be resolved (left). To allow ratcheting by φ29 DNA polymerase (shown in tan), a primer (green fragment) and a blocking oligomer (pink fragment) are required. The blocking oligomer prevents the φ29 polymerase-dependent extension before it reaches the nanopore. The free 5′ end of the polymerase•DNA complex is drawn toward the pore until the polymerase reaches the entrance of the pore (penultimate left). The applied voltage threads the DNA through the pore, causing the blocking oligomer to dissociate (penultimate right). This exposes the 3′ end of the primer and allows synthesis of the complementary strand (shown in yellow, right). The arrows indicate the direction of DNA movement. The position of the narrow constriction is indicated with yellow dots within the nanopore (shown in cyan). Adapted after [125].

Figure 6

Structures of modified (2,3) and unnatural (4–6) nucleotides resolved by nanopore sequencing. 1: cytosine, 2: 5-methylcytosine, 3: 5-hydroxymethylcytosine, 4: dNaM, 5: d5SICS, 6: dTPT3.

Figure 7

Schematic representation of the electrophoretic force and electroosmotic flow (EPF and EOF, respectively) affecting the motion of a negatively charged analyte in a nanopore experiment. Due to the applied voltage, negatively and positively charged ions (shown as red and blue dots, respectively) move toward the electrode of opposite polarity. The charged residues in the pore lumen simultaneously attract counterions and repel coions, creating an imbalance between the positive and negative charge fluxes, resulting in a net water movement and EOF.

Figure 8

ClpX-mediated unfolding promotes protein translocation through a nanopore. A folded protein analyte (pink) is fused to a charged tag (dark blue), and an ssrA tag (bright green). (A) In the absence of ClpX, the charged tag enters the pore lumen; however, the dimensions of the folded protein prevent it from entering the pore. (B) When ClpX (shown in cyan) is present in the trans chamber, it recognizes the protruding ssrA tag and starts to unfold the analyte in an ATP-dependent manner, thereby ensuring translocation of the entire analyte.

Figure 9

The principle of indirect sensing using an aptamer and a competing, complementary DNA probe. (A) In the absence of analyte, the analyte-specific aptamer (in black) forms a duplex with the DNA probe (in green); due to its size, the duplex cannot enter the pore (in grey). (B) When the analyte (purple) is added, the aptamer binds the analyte and the free DNA probe can translocate through the pore. The current signature of DNA probe translocation is unique due to labeling and can be distinguished from other signals generated by nucleic acids present in the sample. The position of the DNA label is indicated by a red circle. The sensing region of the pore is indicated by yellow circles.

Figure 10

Structures of α-HL variants with, β-cyclodextrin (βCD) adapters reported in [178]. α-hemolysin (α-HL) is shown in blue and βCD in red. PDB IDs: 3M2L (M113F₇), 3M3R (M113F₇·βCD), 3M4D (M113N₇), 3M4E (M113N₇·βCD).