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Translocation (nanopore)

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From Molecupedia, the encyclopedia of very small things
Passage of a molecule through a nanopore
For other uses of the word, see Translocation.

Translocation in nanopore sensing is the passage of a molecule from one side of the membrane to the other through the pore, usually driven by an electric field. For a polymer such as DNA it has four stages. In capture the field draws a molecule from the bulk solution toward the pore. Threading begins when one end enters, and during transit the chain is pulled through; at exit the trailing end leaves and the current returns to its open-pore level. Each stage has its own physics and its own duration, and the instrument records the third as an event: a dip in the current, whose depth and width are the current blockade and the dwell time.

The Molecular Reality Corporation guide describes the same trace as "a story in three acts": "First the current drops, as the molecule enters and the doorway narrows. Then a plateau, while the molecule threads through. Then the current springs back as it exits."[1]

Four drawings of a DNA strand and a pore in a membrane, labelled capture, threading, transit and exit, above a current trace that is flat, steps down for the transit and returns; a second trace shows a deeper first part for a folded molecule
The four stages of a translocation, with the current trace each produces. A molecule that enters folded has two strands in the pore at once, and the first part of its event is deeper. A schematic; no measured data.

Capture

Diffusion alone is a poor way to reach a pore. A random walker in three dimensions that starts a distance r from a sphere of radius a ever touches it with probability a/r, so a molecule 100 radii away has a chance of 1%.[2][a] The field changes the odds. Outside a pore of diameter d and length l the potential falls as ΔV d2/(8lr), and a charged molecule with mobility μ drifts along its gradient. Wanunu and colleagues defined a capture radius

r*=d2μ ΔV8 l D

inside which drift outruns diffusion (D is the diffusion coefficient of the molecule). When arrival, rather than threading, limits the rate, the capture rate per unit concentration is 2πDr* = πd2μΔV/(4l), which does not depend on D and therefore not on the length of the molecule.[4] For a thin membrane the length l is replaced by l + πd/4, the correction of Access resistance. Their measurements agreed with this picture above about 8,000 base pairs, where the rate stopped depending on length; below that, shorter molecules were captured less often. A 20-fold salt gradient across the pore raised the sensitivity of the pore more than 30-fold, because it strengthens the field outside, and it also lengthened the translocation times.[4] See Capture rate (nanopore).

Gershow and Golovchenko found that electric forces draw DNA to a pore from micrometer-scale distances, and that a molecule begins to translocate almost immediately on arrival.[5]

Threading

A chain has to find its way in, and for a small pore that is the slow step. In an alpha-hemolysin channel, Henrickson and colleagues found that the rate of blockades is proportional to the concentration of polymer and increases exponentially with the applied potential, the signature of a barrier and not of diffusion; DNA also entered more readily through the end of the channel with the larger vestibule.[6] A double-stranded molecule in a solid-state pore enters by an end, and the entry followed by a complete passage gives the square-shaped pulse that the analysis counts.[4]

Transit

Inside the pore the electric field acts on the charge of the DNA. Keyser and colleagues held a DNA molecule in a solid-state pore with optical tweezers and measured a force of 0.24 pN per mV, independent of salt concentration from 0.02 to 1 M potassium chloride. That corresponds to an effective charge of 0.50 electrons per base pair, a 75% reduction of the bare charge of two per base pair, and to about 48 pN at 200 mV.[7]

The speed that results depends on the pore. In α-hemolysin, polymers longer than the channel cross it at a constant speed, shorter ones faster, and the velocity is nonlinear in the applied field.[8] In 10 nm silicon oxide pores, double-stranded DNA of 6,500 to 97,000 base pairs took a time that grew as the length to the power 1.27, not linearly as in protein pores, and the authors modeled this as the result of hydrodynamic drag on the part of the chain still outside the pore.[9]

Speed of DNA through pores
Condition Speed Source
Solid-state pores, driven by the field alone 10 to 1,000 ns per base (1 to 100 bases per µs) [10]
Same, slowed by temperature, salt, viscosity and voltage 3 bases per µs (3 kbp DNA, 4 to 8 nm silicon nitride pore) [11]
Alpha-hemolysin, driven by the field alone Less than about 1 to 10 µs per base [12]
Alpha-hemolysin, held back by a polymerase 2.5 to 40 nucleotides per second [13]
CsgG pore with a motor, 2016 About 250 bases per second [14]

At those speeds a molecule of lambda phage DNA, 48,502 base pairs and 16.5 µm long, crosses a solid-state pore in 0.5 to 50 ms.

Exit and recapture

The current returns to its open-pore level when the trailing end leaves. A molecule that has just left is still near the pore, and reversing the voltage soon afterward can bring it back so that the same molecule is read again; repeated translocation of one molecule improves the accuracy of the measurement.[5] The practice is called Ping-pong sensing. Two pores a few micrometers apart can pull one molecule in opposite directions and floss it back and forth, and a dual-pore system with that control logic mapped tag positions on fragments of the 4.6 million base pair Escherichia coli chromosome; see Molecular flossing.[15]

Dwell-time distributions

The time a molecule spends in transit is a random variable even when its length is fixed. If the chain moves along the pore axis by drift with speed v and diffusion D, the time to cover a length L has the first-passage distribution of a biased random walk,

f(t)=L4πDt3exp[−(L−vt)24Dt],

an inverse Gaussian with mean L/v and variance 2DL/v3.[2] Gershow and Golovchenko found that the rates and times of recapture agreed with solutions of a drift-diffusion model.[5] The chain as a whole has its own theory, from Muthukumar's and Lubensky and Nelson's models of 1999 to a review of its first two decades in 2014.[16][17][18]

Events shorter than the response time of the electronics never appear as events. For proteins in solid-state pores, Plesa and colleagues found event rates orders of magnitude below what diffusion predicts, and a one-dimensional first-passage model suggested that most of the molecules translocate faster than can be detected.[19]

Folds and knots

Long DNA does not always enter by an end. Li and colleagues, who built a "solid-state nanopore microscope" in 2003, watched double-stranded DNA fold, and Storm and colleagues sorted molecules of 6,557 to 48,500 base pairs by whether they crossed straight or folded; circular molecules, which must cross doubled, supported the picture.[20][21] A fold puts two strands in the pore at once, and in a pore wide enough for both the current is blocked about twice as far for the folded part of the event. Glass nanocapillaries read the folding state of lambda DNA in the same way.[22] Knots show as short spikes on the trace. In 2016 Plesa and colleagues observed them in linear and circular molecules up to 166,000 base pairs long and estimated from the transit time of the knot that most were tight, under 100 nm across.[23] The company's demonstration of the same signal drew a knotted nylon filament through a pore about 2 mm wide and recorded "downward (resistive) spikes".[24] See Knot sequencing.

Too fast to read

Deamer, Akeson and Branton wrote that even the smallest bias that drives DNA through a pore moves each base through the sensing region in under about 1 to 10 µs, and that identifying a base from picoampere currents needs it to stay for more than 100 µs.[12] In a solid-state pore a base driven by the field alone passes 100 to 10,000 times faster than the 100 µs that reading needs. Rosenstein and colleagues, who recorded at 1 MHz with a signal-to-noise ratio above 5, observed that nanopore measurements had been constrained below 100 kHz by noise.[10] A later amplifier reached 5 MHz with a signal-to-noise ratio above 10 and saw transient features 200 ns long.[25]

The company calls the mismatch the "Speeding Bullet" problem and describes DNA as translocating "far too rapidly" for electronics to resolve single bases.[26] Its 2022 overview puts the consequence plainly: "Without translocation control, analytes usually move too quickly through a pore, so the electronics can't integrate enough current data to tell anything about them or even, in some cases, know that anything went through at all."[27] Three remedies have been tried. Fologea and colleagues changed the temperature, salt, viscosity and voltage of the whole system. For molybdenum disulfide pores, whose DNA speeds Feng and colleagues give as 3,000 to 50,000 nucleotides per millisecond, they used a viscosity gradient built from room-temperature ionic liquids.[28] A motor protein advances the strand by chemistry instead of by the field and brings the speed to hundreds of bases per second. The company's proposal is geometric, and the guide puts it in one line: "reading requires slowing the molecule down, and slowing molecules down is what sliding occlusion is for."[1] See Speeding bullet problem, Mechanical nanopore and Electrophoresis.

See also

Notes

  1. ^ That a three-dimensional walk can wander off for good is Pólya's theorem of 1921. The chance that a walk on a cubic lattice ever returns to its starting point is 0.34.[3]

References

  1. ^ a b "Keltar's Field Guide to Molecular Reality". Molecular Reality Corporation. August 2026.
  2. ^ a b Redner, S. (2001). A Guide to First-Passage Processes. Cambridge University Press. ISBN 978-0-521-65248-5.
  3. ^ Pólya, G. (1921). "Über eine Aufgabe der Wahrscheinlichkeitsrechnung betreffend die Irrfahrt im Straßennetz". Mathematische Annalen 84 (1-2): 149-160. doi:10.1007/BF01458701.
  4. ^ a b c Wanunu, M.; et al. (2010). "Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient". Nature Nanotechnology 5 (2): 160-165. doi:10.1038/nnano.2009.379. PMID 20023645.
  5. ^ a b c Gershow, M.; Golovchenko, J. A. (2007). "Recapturing and trapping single molecules with a solid-state nanopore". Nature Nanotechnology 2 (12): 775-779. doi:10.1038/nnano.2007.381. PMID 18654430.
  6. ^ Henrickson, S. E.; et al. (2000). "Driven DNA transport into an asymmetric nanometer-scale pore". Physical Review Letters 85 (14): 3057-3060. doi:10.1103/PhysRevLett.85.3057.
  7. ^ Keyser, U. F.; et al. (2006). "Direct force measurements on DNA in a solid-state nanopore". Nature Physics 2 (7): 473-477. doi:10.1038/nphys344.
  8. ^ Meller, A.; Nivon, L.; Branton, D. (2001). "Voltage-driven DNA translocations through a nanopore". Physical Review Letters 86 (15): 3435-3438. doi:10.1103/PhysRevLett.86.3435.
  9. ^ Storm, A. J.; Storm, C.; Chen, J.; Zandbergen, H.; Joanny, J. F.; Dekker, C. (2005). "Fast DNA translocation through a solid-state nanopore". Nano Letters 5 (7): 1193-1197. doi:10.1021/nl048030d.
  10. ^ a b Rosenstein, J. K.; et al. (2012). "Integrated nanopore sensing platform with sub-microsecond temporal resolution". Nature Methods 9 (5): 487-492. doi:10.1038/nmeth.1932. PMID 22426489.
  11. ^ Fologea, D.; et al. (2005). "Slowing DNA translocation in a solid-state nanopore". Nano Letters 5 (9): 1734-1737. doi:10.1021/nl051063o.
  12. ^ a b Deamer, D.; Akeson, M.; Branton, D. (2016). "Three decades of nanopore sequencing". Nature Biotechnology 34 (5): 518-524. doi:10.1038/nbt.3423. PMID 27153285.
  13. ^ Cherf, G. M.; et al. (2012). "Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision". Nature Biotechnology 30 (4): 344-348. doi:10.1038/nbt.2147.
  14. ^ "Update: New "R9" nanopore for faster, more accurate sequencing, and new ten minute preparation kit". Oxford Nanopore Technologies. May 26, 2016. Retrieved October 25, 2026.
  15. ^ Rand, A.; et al. (2022). "Electronic mapping of a bacterial genome with dual solid-state nanopores and active single-molecule control". ACS Nano 16 (4): 5258-5273. doi:10.1021/acsnano.1c09575. PMID 35302746.
  16. ^ Muthukumar, M. (1999). "Polymer translocation through a hole". The Journal of Chemical Physics 111 (22): 10371-10374. doi:10.1063/1.480386.
  17. ^ Lubensky, D. K.; Nelson, D. R. (1999). "Driven polymer translocation through a narrow pore". Biophysical Journal 77 (4): 1824-1838. doi:10.1016/S0006-3495(99)77027-X.
  18. ^ Palyulin, V. V.; Ala-Nissila, T.; Metzler, R. (2014). "Polymer translocation: the first two decades and the recent diversification". Soft Matter 10 (45): 9016-9037. doi:10.1039/c4sm01819b.
  19. ^ Plesa, C.; et al. (2013). "Fast translocation of proteins through solid state nanopores". Nano Letters 13 (2): 658-663. doi:10.1021/nl3042678.
  20. ^ Li, J.; et al. (2003). "DNA molecules and configurations in a solid-state nanopore microscope". Nature Materials 2 (9): 611-615. doi:10.1038/nmat965.
  21. ^ Storm, A. J.; Chen, J. H.; Zandbergen, H. W.; Dekker, C. (2005). "Translocation of double-strand DNA through a silicon oxide nanopore". Physical Review E 71 (5): 051903. doi:10.1103/PhysRevE.71.051903.
  22. ^ Steinbock, L. J.; et al. (2010). "Detecting DNA folding with nanocapillaries". Nano Letters 10 (7): 2493-2497. doi:10.1021/nl100997s.
  23. ^ Plesa, C.; et al. (2016). "Direct observation of DNA knots using a solid-state nanopore". Nature Nanotechnology 11 (12): 1093-1097. doi:10.1038/nnano.2016.153.
  24. ^ Kemmishtree (March 29, 2026). "Sequencing a string". EQBio-OS.
  25. ^ Shekar, S.; et al. (2016). "Measurement of DNA translocation dynamics in a solid-state nanopore at 100 ns temporal resolution". Nano Letters 16 (7): 4483-4489. doi:10.1021/acs.nanolett.6b01661.
  26. ^ "D&D: Disclosure Crawl". Molecular Reality Corporation.
  27. ^ "Scientific and technical overview". Molecular Reality Corporation. December 2022.
  28. ^ Feng, J.; et al. (2015). "Identification of single nucleotides in MoS2 nanopores". Nature Nanotechnology 10 (12): 1070-1076. doi:10.1038/nnano.2015.219. PMID 26389660.
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