In 2014, Kim and co-workers demonstrated a single-molecule DNA sequencing concept in which a DNA polymerase and its primer-template, co-immobilized on an atomic force microscope (AFM) tip, sequentially recognize which of four surface-tethered nucleotides is complementary to the template base in the active site, after which the identified base is incorporated and the next base becomes readable; a single template was read contiguously for 11 bases. The study explicitly identified the obstacles separating this proof of principle from a practical targeted single-DNA sequencing method, and none has since been overcome: functional tip yield of only about 20% with uncontrolled polymerase orientation; read length bounded by the elasticity of the primer-tip spacer and cumulative tip contacts (a projection of roughly 40 bases with optimized linkers); on-chip incorporation of surface-tethered nucleotides demonstrated only for a bulky gamma-phosphate-biotin dGTP analog and only at an applied normal force of 0 pN, with incorporation absent at applied loads of plus or minus 10 to 20 pN; the unanswered mechanistic and engineering question of whether polymerases can be engineered or selected for reliable catalytic activity in defined force regimes; and unaddressed throughput and scalability.

Background

Reading the base sequence of a single DNA molecule without amplification is a long-standing goal of single-molecule biophysics, and by 2014 two routes had reached practice: sequencing by synthesis in zero-mode-waveguide fluorescence (commercialized by Pacific Biosciences) and, emerging then, nanopore ionic-current sequencing. Kim, Lee, Park and co-workers proposed a third, mechanical route built on the atomic force microscope (AFM), an instrument in which a sharp tip on a flexible cantilever is pressed against a surface so that piconewton-scale forces bend the cantilever; in single-molecule force spectroscopy, a molecule tethered to the tip and a partner molecule tethered to the surface are pulled apart, and the force at which the specific complex ruptures (the rupture force) is measured, while force mapping raster-scans the tip over the surface to map where such specific interactions occur.

In their scheme (Kim et al., J. Am. Chem. Soc. 2014, doi:10.1021/ja5063983), the enzyme is Therminator gamma DNA polymerase, an archaeal 9 degrees N polymerase variant engineered to accept chemically modified nucleotides; it is co-immobilized on the AFM tip together with its primer-template DNA (a short primer hybridized to the template to be read). The four deoxynucleoside triphosphates (dNTPs, the monomeric substrates of DNA synthesis) are tethered in separate patches on a surface. When the tip-borne polymerase contacts the patch containing the nucleotide complementary to the template base currently occupying its active site, it binds that nucleotide specifically, producing a characteristic rupture event of about 26-32 pN upon tip retraction; the other three patches give no such signal. After the complementary base is thus identified, it is added in solution so the polymerase incorporates it and the next template base enters the active site, and the cycle repeats. Alternating recognition and in-solution incorporation, the authors read 11 consecutive bases of one template molecule. They additionally showed on-chip incorporation, in which the polymerase incorporates the surface-tethered nucleotide directly during AFM contact in force-clamp mode (a feedback mode that holds the tip-sample force constant): ten 10-second contacts at an applied normal force of 0 pN0\ \mathrm{pN} incorporated a streptavidin-bound γ\gamma-phosphate-biotin dGTP analog, whereas compressive loads of −10-10 and −20 pN-20\ \mathrm{pN} and tensile loads of 1010 and 20 pN20\ \mathrm{pN} prevented incorporation.

The mechanics of polymerase catalysis under load was not new territory, but the established results concern a different geometry: Wuite et al. (Nature 2000, doi:10.1038/35003614) showed with optical tweezers that T7 DNA polymerase replicates against tension applied to the DNA template itself, working against up to roughly 34 pN with a tension-dependent rate, and theoretical work by Xie (J. Mol. Model. 2013, doi:10.1007/s00894-012-1688-5) predicted that a backward force applied to the Klenow-fragment enzyme has a large, non-monotonic effect on replication velocity, including an optimal backward force. None of that concerns a normal load applied to a surface-engaged enzyme as it incorporates a surface-tethered, chemically modified nucleotide, the configuration the AFM scheme requires. Later AFM work stayed orthogonal: Chao et al. (Nanoscale 2016, doi:10.1039/c5nr06544e) imaged Klenow-fragment polymerase activity on DNA by AFM without force-controlled incorporation, and the original group itself moved to force-based AFM quantification of nucleic acid biomarkers and DNA-ligation nanopatterning (e.g., Kim et al., Nanomaterials 2021, doi:10.3390/nano11071725). What the 2014 paper left unresolved is therefore still unresolved: the conditions, chemistry, and enzyme engineering under which polymerase-catalyzed phosphodiester bond formation operates reliably under AFM force control, and the probe, linker, and throughput engineering that would turn the demonstrated 11-base read into a practical method.

Problem Statement

The problem, as posed by the source study (Kim et al., J. Am. Chem. Soc. 2014, doi:10.1021/ja5063983), is to advance the AFM-based read-then-incorporate workflow for sequencing a single DNA molecule from its 11-base proof of principle toward a practical targeted single-DNA sequencing method, by resolving the obstacles the authors identified. These obstacles stand between the demonstrated proof of principle and such a practical method:

  1. Tip functionalization: raise the functional yield of polymerase/primer co-immobilized AFM tips (about 20% in tested preparations) and control polymerase orientation during immobilization.

  2. Read length: overcome the dominant limit, the elasticity and extensibility of the primer-tip spacer as the primer lengthens with each incorporated base, together with cumulative tip damage from repeated contacts, toward reliably extended contiguous reads (the authors project up to roughly 40 bases with suitable linkers, a projection that itself rests on untested assumptions about tip integrity and linker behavior).

  3. On-chip incorporation chemistry: extend direct incorporation of surface-tethered nucleotides beyond the single bulky γ\gamma-phosphate-biotin dGTP analog demonstrated, while suppressing unwanted incorporation during the recognition step through control of contact time and applied force.

  4. Force compatibility of catalysis: the observed strong dependence of polymerase catalytic competence on applied mechanical force, with on-chip incorporation at 0 pN0\ \mathrm{pN} normal force but not under modest compressive or tensile loads (±10\pm 10-20 pN20\ \mathrm{pN}), raises the open question of whether polymerases can be engineered or selected to have enhanced, reliable catalytic activity in reproducible force regimes, so that on-chip chemistry proceeds robustly under AFM force control.

  5. Throughput and scalability: achieve the throughput and scalability required for the method to support practical targeted single-DNA sequencing.

The obstacles to be resolved are the five listed above. The pivotal scientific unknown is the pair (3)-(4): whether polymerase-catalyzed phosphodiester bond formation can be made to operate reliably under a defined mechanical load with surface-tethered nucleotide substrates. Items (1), (2), and (5) are the coupled engineering conditions under which such force-compatible catalysis would translate into extended, reliable single-molecule reads.

References

  1. Youngkyu Kim, Eung-Sam Kim, Yoonhee Lee, Joung-Hun Kim, Bong Chu Shim, Seong Moon Cho, Jeong Soo Lee, Joon Won Park, "Reading Single DNA with DNA Polymerase Followed by Atomic Force Microscopy", Journal of the American Chemical Society 136(39), 13754-13760 (2014). doi:10.1021/ja5063983, https://doi.org/10.1021/ja5063983 (primary source)
  2. G. J. L. Wuite, S. B. Smith, M. Young, D. Keller, C. Bustamante, "Single-molecule studies of the effect of template tension on T7 DNA polymerase activity", Nature 404(6773), 103-106 (2000). doi:10.1038/35003614, https://doi.org/10.1038/35003614
  3. P. Xie, "Dynamics of DNA polymerase I (Klenow fragment) under external force", Journal of Molecular Modeling 19(3), 1379-1389 (2013). doi:10.1007/s00894-012-1688-5, https://doi.org/10.1007/s00894-012-1688-5
  4. J. Chao, P. Zhang, Q. Wang, N. Wu, F. Zhang, J. Hu, C. H. Fan, B. Li, "Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy", Nanoscale 8, 5842-5846 (2016). doi:10.1039/c5nr06544e, https://doi.org/10.1039/c5nr06544e
  5. Youngkyu Kim, Woong Kim, Joon Won Park, "Principles and Applications of Force Spectroscopy Using Atomic Force Microscopy", Bulletin of the Korean Chemical Society 37, 1895-1907 (2016). doi:10.1002/bkcs.11022, https://doi.org/10.1002/bkcs.11022
  6. Eung-Sam Kim, Jung Sook Kim, Nishan Chakrabarty, Chul-Ho Yun, "Covalent Positioning of Single DNA Molecules for Nanopatterning", Nanomaterials 11(7), 1725 (2021). doi:10.3390/nano11071725, https://doi.org/10.3390/nano11071725
  7. Jinxin Xiong, Zhimei He, Lianhui Wang, Chunhai Fan, Jie Chao, "DNA Origami-Enabled Gene Localization of Repetitive Sequences", Journal of the American Chemical Society 146(9), 6317-6325 (2024). doi:10.1021/jacs.4c00039, https://doi.org/10.1021/jacs.4c00039