Figure 1. Schematic illustration of the basic concepts and principles of nanopore DNA sequencing. (a) Schematic diagram of the nanopore single-molecule sequencing platform. The electrolyte solution (usually KCl buffer) is injected into the measurement tank, and the tank is divided into two chambers by a Teflon film. There is a support pore with a diameter of approximately 100 μm in the middle of the film, which is used to bind the phospholipid bilayer. Subsequently, biological nanopores are added in the solution to spontaneously insert into the phospholipid layer, forming a nanoscale measurement channel[86]. Phospholipid membrane is shown in gray, and the biological nanopores are presented as blue channels. (b) Schematic diagram of the structure and sensing area of MspA nanopores. A pair of electrodes is inserted into the two sides of the phospholipid bilayer, with a negative voltage on the cis side and a positive voltage on the trans side. I0 represents the ion current within the nanopores when no analyte is perforated. (c) Schematic of MspA nanopore sequencing. Single-stranded DNA (ssDNA) forms a complex with the DNA polymerase phi29, and this complex can stably remain at the nanopore port. Under the action of polymerase, ssDNA begins to synthesize the double helix structure. During this process, the primer extension "force" of the polymerase drives the stepped-translocation of ssDNA within the nanopores, pulling out the ssDNA from the nanopores one base at a time. When ssDNA molecules enter the sensing region, they block the nano-channels, reducing the ionic current and thereby generating the blocking current signal Ib. (d) Nanopore blockade current and sequencing. When ssDNA molecules with different sequences pass through nanopores, blocking currents with different durations and amplitudes are generated[31]. By analyzing the depth and time of blockade currents, DNA can be directly sensed at the single-molecule level, and the DNA sequence information can be analyzed through base calling algorithms.

Figure 2. Discovery, structure, and key mutation sites of five biological nanopores. From left to right: schematic diagrams of the structure and key mutation sites of α-HL, Aerolysin, MspA, FraC, and CsgG nanopores. Protein backbones of these nanopores are shown in grey, amino acids at key mutation sites are shown as van der Waals surfaces, and the sensing regions are presented with Green transparent areas. Protein structures in the schematic diagram are from RCSB PDB database. α-HL nanopores are the earliest biological nanopores to be applied in DNA single-molecule measurement[24], and the spatial resolution of their sensing regions can distinguish between purine and pyridine fragments. Aerolysin nanopores have long and narrow sensing regions similar to α-HL nanopores. Compared with α-HL nanopores, the sensing regions of Aerolysin nanopores have more key sites, which is conducive to the construction of diverse functional channels[60]. Compared with the former two, MspA nanopores exhibit high-resolution advantages in DNA sequencing due to their shorter and narrower single-molecule sensing regions[31]. Frac nanopores are characterized by their α-helix-typed transmembrane domains rather than β-folding structures[77], which endows the Frac nanopores with tunable pore sizes. CsgG nanopores have been developed and applied to the commercial DNA sequencing platform of ONT. They can form CsgG-CsgF composite nanopores with CsgF[85], and this kind of composite nanopores has a secondary sensing region to enhance the sensing performance.

Figure 3. Schematic diagram of motor proteins controlling the kinetics of DNA translocation within nanopores. MspA nanopores are presented in blue, and motor proteins are shown in yellow. (a) phi29 speed control mechanism and characteristics: Through polymerization reaction, phi29 gradually pulls the single strand of the DNA translocating through the nanopore while synthesizing the double strands of DNA. The arrow indicates the movement direction of the DNA. (b) Hel308 speed control mechanism and characteristics: Through the ATP hydrolysis cycle, Hel308 step by step unties the DNA double helix, allowing the DNA translocation in the nanopore. The arrow indicates the movement direction of the DNA.

Figure 4. The key factors affecting the sequencing accuracy and efficiency of nanopore single-molecule platforms include: Sequencing spatial resolution optimization based on nanopore structure modification (top, mutant CytK nanopore, protein structure data from RCSB PDB website, https://www.rcsb.org), DNA translocation dynamics control and sequencing time resolution optimization based on motor proteins (middle left, phi29 polymerase-DNA complex, structural model data from Alamy website, https://www.alamy.com), base calling algorithm and sequencing program (middle right, schematic diagram of base calling algorithm flow), high-throughput sequencing technology based on the parallel operation of multi-channel fluid channels (lower left, high-throughput sequencing system schematic diagram) and integrated sequencing system based on in-chip architecture (lower right, integrated nanopore sequencing platform).

Figure 5. The application scenarios of nanopore DNA sequencing technology include: genomics research, precision medicine and epidemic supervision, and real-time on-site sequencing. The schematic diagrams of genomic research and the sequence diagrams of the novel coronavirus are respectively cited from references [137] and [143] and have been granted permission. The schematic diagram of the space station's sequencing is from the news photo at www.nasaspaceflight.com.