Jump to content
[dismiss]
Molecupedia is a parody encyclopedia written by Molecular Reality Corporation. It is not Wikipedia and is not affiliated with the Wikimedia Foundation. About

Motor protein (nanopore)

Page protected
From Molecupedia, the encyclopedia of very small things
Enzyme that moves a DNA, RNA or protein strand through a nanopore one step at a time

In nanopore sequencing, a motor protein is an enzyme that grips a strand at the mouth of a pore and feeds it through one step at a time. A strand pulled through a pore by the electric field alone passes each base in a microsecond or less, too fast for the current to be read. An enzyme that holds the strand, advances it by one nucleotide and holds it again slows the passage to milliseconds per base. Polymerases and helicases do this for DNA, and an unfoldase does it for proteins. The sequencers of Oxford Nanopore Technologies use one, and it is what slows the strand enough for a protein pore to read it (see speeding bullet problem).[1]

Principle

A protein pore with two reader heads in a membrane, a motor protein at its mouth and a strand of colored bases passing through, beside a staircase current trace of eight levels between about 60 and 120 picoamperes
Strand sequencing with a motor protein, schematic. The enzyme sits above the pore and lets the strand through one base at a time; each step shows as a new current level. The trace is illustrative: eight levels of 2.5 ms each, a rate of 400 bases per second, with noise added.

A strand carries the negative charge of its phosphate backbone, so the field pulls it toward the positive electrode. Because the pore is too narrow for the enzyme, the enzyme stays on the entry side and holds the strand while the field tugs on the part inside the pore. Each completed cycle of the enzyme lets the strand move by one unit, and in the current the steps appear as stairs. The enzyme sets the step: a phi29 DNA polymerase ratcheted DNA through alpha-hemolysin with a precision of 5 Å, and the helicase Hel308 pulls DNA through MspA in steps of about 0.33 nm, half a nucleotide.[2][3]

Speeds follow from the chemistry of the enzyme: the polymerase in the 2012 experiments moved DNA at median rates of 2.5 to 40 nucleotides per second, and Oxford Nanopore says that its flow cells have run at about 400 to 450 bases per second since October 2016, which is 2.2 to 2.5 ms for each base.[2][4] The enzyme needs fuel: a polymerase consumes deoxynucleoside triphosphates as it copies the strand, and a helicase consumes ATP.

Why a slow strand is a quiet one

The motor helps the noise as well as the clock. An amplifier needs a bandwidth of about 0.35 divided by the duration of an event to follow it, so a base that lasts 1 µs calls for 350 kHz and one that lasts 2.5 ms calls for 140 Hz. The thermal noise current of a given feedback resistor grows as the square root of the bandwidth (see Johnson-Nyquist noise), so the slower strand can be read through a band 2,500 times narrower, which carries 50 times less of that noise.[a]

Enzymes used

Polymerases came first. The bacteriophage phi29 DNA polymerase has "remarkable processivity and high affinity for DNA substrates", and it stays bound to DNA inside the electric field across a pore.[5] Helicases are the second family. The Hel308 helicase pulls DNA through MspA, and the same enzyme gave the peptide rereads described below. For proteins, the AAA+ unfoldase ClpX, a bacterial enzyme that unfolds proteins and threads them into a protease, drove proteins through alpha-hemolysin in 2013 and, in 2024, through the CsgG pore of a commercial sensor array, where it translocated in two-residue steps.[6][7]

History

Enzymes and nanopores
Year Result
2007 A nanopore told apart unbound DNA, DNA bound to a polymerase and DNA bound to a polymerase and a nucleotide, in real time[8]
2008 Up to nine consecutive single-nucleotide extensions by a DNA polymerase, detected with a spatial resolution of 2.4 Å or better in a strand held in alpha-hemolysin[9]
2009 A pore with a covalent adapter identified unlabelled nucleoside monophosphates, the products an exonuclease would release, one at a time with an average accuracy of 99.8%[10]
2010 Sequential nucleotide additions by phi29 DNA polymerase detected in real time as the template was drawn through alpha-hemolysin[5]
2012 Two papers in one issue of Nature Biotechnology: phi29 ratcheting DNA through alpha-hemolysin, and phi29 pulling it through MspA with levels lasting a median of 28 ms[2][11]
2013 ClpX drives proteins through alpha-hemolysin[6]
2015 Hel308 followed along DNA with about 40 pm sensitivity[12]
2021 A single peptide pulled back and forth through MspA by Hel308 and read 117 times[3]
2024 ClpX pushes protein strands hundreds of amino acids long through CsgG on a commercial sensor array[7]

Limits

A motor sets the speed, and the steps it takes are not uniform. In the 2012 experiments, without optimization, the probability of a registry error, an inserted or skipped position, in a single pass along a template ranged from 10% to 24.5%.[2] Because the reads can be rewound, one peptide was read 117 times in 2021, and above about 30 rereads the error rate for single amino acid variants fell below one in a million.[3] Ping-pong sensing and molecular flossing reread a molecule without an enzyme.

Without an enzyme

Lithium chloride in place of potassium chloride slows DNA in a solid-state pore.[13] Two pores that pull a strand against each other slowed it by up to two orders of magnitude, and a strand confined in a zeptoliter water bridge between two pores 20 nm apart was slowed by up to three.[14][15] No laboratory has published a solid-state pore reading DNA base by base, and the founder's 2022 overview of the field says the same.[16]

At Molecular Reality

Molecular Reality Corporation works with solid-state nanopores, which no enzyme drives. Its founder has written about the problem that the enzymes solve. In a post on a paper that tethered single proteins to a lipid coating inside a solid-state pore, he drew the lesson that "translocation control is most powerful when degrees of freedom are reduced".[17] His post on the zeptoliter bridge calls the arrangement "a form of translocation control", and says the strand "gets slowed down significantly by friction with the dividing wall between the two pores".[18]

The company's own proposal, a pore whose opening can be narrowed around a molecule, is described in the article on sliding occlusion.

See also

Notes

  1. ^ Calculated: 350 kHz ÷ 140 Hz = 2,500, and √2,500 = 50. The rule of thumb for the rise time of a first-order filter is bandwidth = 0.35/duration; real recordings are filtered higher than this minimum.

References

  1. ^ Deamer, D.; Akeson, M.; Branton, D. (2016). "Three decades of nanopore sequencing". Nature Biotechnology 34 (5): 518-524. doi:10.1038/nbt.3423.
  2. ^ a b c d 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.
  3. ^ a b c Brinkerhoff, H.; Kang, A. S. W.; Liu, J.; Aksimentiev, A.; Dekker, C. (2021). "Multiple rereads of single proteins at single-amino acid resolution using nanopores". Science 374 (6574): 1509-1513. doi:10.1126/science.abl4381.
  4. ^ "Continuous development and improvement". Oxford Nanopore Technologies. Retrieved October 25, 2026.
  5. ^ a b Lieberman, K. R.; et al. (2010). "Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase". Journal of the American Chemical Society 132 (50): 17961-17972. doi:10.1021/ja1087612.
  6. ^ a b Nivala, J.; Marks, D. B.; Akeson, M. (2013). "Unfoldase-mediated protein translocation through an α-hemolysin nanopore". Nature Biotechnology 31 (3): 247-250. doi:10.1038/nbt.2503.
  7. ^ a b Motone, K.; et al. (2024). "Multi-pass, single-molecule nanopore reading of long protein strands". Nature 633 (8030): 662-669. doi:10.1038/s41586-024-07935-7.
  8. ^ Benner, S.; et al. (2007). "Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore". Nature Nanotechnology 2 (11): 718-724. doi:10.1038/nnano.2007.344.
  9. ^ Cockroft, S. L.; et al. (2008). "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution". Journal of the American Chemical Society 130 (3): 818-820. doi:10.1021/ja077082c.
  10. ^ Clarke, J.; et al. (2009). "Continuous base identification for single-molecule nanopore DNA sequencing". Nature Nanotechnology 4 (4): 265-270. doi:10.1038/nnano.2009.12.
  11. ^ Manrao, E. A.; et al. (2012). "Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase". Nature Biotechnology 30 (4): 349-353. doi:10.1038/nbt.2171.
  12. ^ Derrington, I. M.; et al. (2015). "Subangstrom single-molecule measurements of motor proteins using a nanopore". Nature Biotechnology 33 (10): 1073-1075. doi:10.1038/nbt.3357.
  13. ^ Kowalczyk, S. W.; et al. (2012). "Slowing down DNA translocation through a nanopore in lithium chloride". Nano Letters 12 (2): 1038-1044. doi:10.1021/nl204273h.
  14. ^ Liu, X.; et al. (2019). "Controlling DNA tug-of-war in a dual nanopore device". Small 15 (30): 1901704. doi:10.1002/smll.201901704.
  15. ^ Cadinu, P.; et al. (2017). "Single molecule trapping and sensing using dual nanopores separated by a zeptoliter nanobridge". Nano Letters 17 (10): 6376-6384. doi:10.1021/acs.nanolett.7b03196.
  16. ^ Kemmishtree (March 28, 2026). "2022 sci-tech overview". EQBio-OS.
  17. ^ Kemmishtree (March 29, 2026). "The Zeptoliter Strikes Again". EQBio-OS.
  18. ^ Kemmishtree (March 29, 2026). "Zeptoliter is My Favorite Unit of Measurement". EQBio-OS.
Retrieved from "https://molecularreality.com/wiki/Motor_protein_(nanopore)"
Last edited 1 hour ago by Aperture tube