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DNA

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From Molecupedia, the encyclopedia of very small things
Polymer of nucleotides that carries genetic information, as a physical object

DNA (deoxyribonucleic acid) is a polymer of four kinds of nucleotide that in nearly all cells forms a double helix and carries the cell's genetic information in the order of its units. This article is about the molecule as a physical object, which is how a nanopore meets it: a rod 2 nm across, as long as meters in a human cell, carrying a negative charge on its outside and bending like a thread. The measurements that matter for a pore are its diameter, its length, its stiffness, its charge and what the water and the salt around it do to that charge.

A nanopore turns these into a current. A strand pulled through the narrowest part of a pore displaces salt water, and the ionic current falls, or at low salt rises, while it is there.

A circle 2 nm across for double-stranded DNA drawn beside circles for pores of 1.5, 2.0 and 6.4 nm
The cross-section of double-stranded DNA drawn to the same scale as three pores: the 1.5 nm channel of α-hemolysin, a pore of 2.0 nm made by breakdown, and one of 6.4 nm. A drawing.

Structure

The model of Watson and Crick of 1953 put the sugar-phosphate chains on the outside of the helix and the bases inside. It placed a phosphorus atom 10 Å from the fibre axis, a residue on each chain every 3.4 Å along the axis, and ten residues per turn, so that the structure repeats after 34 Å.[1] The paper ends on a sentence that is quoted more often than its numbers: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." The diameter of the helix is therefore about 2 nm, a base pair adds 0.34 nm to the length, and a turn is 3.4 nm long. A pore sees a little more than the model does, because the hydrated molecule is slightly wider; a paper on making pores used double-stranded DNA of "~2.2 nm in diameter" as a ruler for their size.[2]

Lengths follow from the rise per base pair. The bacteriophage lambda DNA, a common test molecule in nanopore laboratories, has 48,502 base pairs and is 16.5 µm long.[3][4] A human cell holds more. The diploid nuclear genome of a man has 6.27 billion base pairs and is 205 cm long, and that of a woman has 6.37 billion and 208 cm, so that about two meters of DNA lie in each nucleus.[5]

Stiffness

DNA is neither a rigid rod nor a limp string. Its persistence length, the distance over which a straight stretch forgets its direction, is about 150 base pairs, or 51 nm, the value used in the theory of DNA capture.[3] A molecule much longer than that coils. Lambda DNA, 16.5 µm long, is about 320 persistence lengths, and the worm-like chain model puts the radius of gyration of such a coil at about 0.5 µm.[a] Such a coil is about a micrometer wide, some 200 times the width of a pore of 5 nm, and the pore must catch one end of it. How that happens is described in Capture rate (nanopore), and what the molecule does inside in Translocation (nanopore), where a chain that enters folded makes a blockade about twice as deep as one that enters by an end.[7]

Single strands go through narrower openings. The 1.5 nm channel of α-hemolysin admits single strands, and the first experiment that drove DNA through a pore used it.[8] Pores of 2.0 and 2.1 nm made by controlled breakdown have been able to tell single-stranded from double-stranded DNA by the depth of the blockade.[9]

Charge

Each phosphate in the backbone carries one negative charge, and each base pair therefore has two, about 5.9 per nanometer of length. Positive ions from the solution gather around the strand, and the Manning theory of counterion condensation, published in 1969, accounts for the fact that a large part of the charge is neutralized this way.[10] In 2006 DNA held in a solid-state pore by optical tweezers was found to feel a force of 0.24 ± 0.02 pN for each millivolt applied across the membrane, independent of the salt concentration from 0.02 to 1 M potassium chloride. That corresponds to an effective charge of 0.50 ± 0.05 electrons per base pair, a reduction of 75% from the bare charge, and to about 48 pN at 200 mV.[11]

In free solution the mobility of DNA does not depend on its length, because charge and drag both grow with the number of units. The diffusion-limited capture rate depends on the mobility and not on the diffusion coefficient, so it does not depend on length either.[3] At the mouth of a pore the balance changes: a coil pressed against the membrane has no electro-osmotic flow through it, and its effective charge grows in proportion to its length.[3]

Sequence

The order of the four bases is the information. A nanopore sequencer feeds a single strand through a pore and reads the order from the current, which depends on the few bases in the narrowest part of the pore at a time. In the maker's public table of expected currents for the R9.4 pore, the 4,096 six-base words run from 58.5 to 119.7 pA, the word AAAAAA has a level of 86.5 pA, and changing its last base to C lowers the level to 83.9 pA.[12] Turning a series of such levels into letters is the subject of Basecalling.

What a pore measures

The strand blocks current by excluding salt water. In a pore 6.4 nm wide in a film 10 nm thick, with the DNA in 3.6 M lithium chloride at 200 mV, the single-level blockades of 5-kilobase DNA were 7.4 ± 0.9 nS in conductance, which with a DNA diameter of 2.2 nm gives a pore of 6.0 ± 0.5 nm, near the 6.4 nm that the conductance of the empty pore gave.[2]

The counterions complicate the picture. For double-stranded DNA 16.5 µm long, translocation decreased the ionic current when the potassium chloride was stronger than 0.4 M and increased it when it was weaker, and the two effects cancelled at 370 ± 40 mM. The authors attributed the decreases to the partial blocking of the pore and the increases to the motion of the counterions that screen the charge of the backbone.[4] The ions travel with the strand, so the strand adds conductance as well as removing it.

Speed sets what can be read. Driven by the field alone, DNA crosses a solid-state pore at 10 to 1,000 ns per base,[13] which is 0.34 to 34 mm a second along the backbone, and a lambda molecule goes through in half a millisecond to 50 ms; see Speeding bullet problem. In a sequencer a motor protein holds the speed at hundreds of bases a second, and the sequence is recovered from the current by basecalling; see Nanopore sequencing.

At Molecular Reality

Molecular Reality Corporation publishes a video of "Fields of DNA molecules at 100×" of February 19, 2019, in which single DNA molecules under fluorescence are moved by electric fields,[14] and its records list the mechanical cleavage of DNA ropes in the same month.[15] The company's Church of Sequence Space promotes "understanding of aperiodic polymers like DNA as the key to solving biology to free our species from disease".[15] The phrase echoes the "aperiodic crystal" that Erwin Schrödinger gave in 1944 as the nature of the hereditary material; see Aperiodic crystal.[16] Its machine-learning work uses recordings made by the Keyser group at the University of Cambridge of DNA carriers that bear a three-bit barcode of small hairpins; see DNA barcode (nanopore) and QuipuNet.[17]

See also

Notes

  1. ^ For a chain much longer than its persistence length P, the mean square end-to-end distance is 2PL and the mean square radius of gyration PL/3. With P = 51 nm and L = 16.5 µm the radius of gyration is 0.53 µm.[6]

References

  1. ^ Watson, J. D.; Crick, F. H. C. (1953). "Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid". Nature 171 (4356): 737-738. doi:10.1038/171737a0.
  2. ^ a b Kwok, H.; Briggs, K.; Tabard-Cossa, V. (2014). "Nanopore fabrication by controlled dielectric breakdown". PLOS ONE 9 (3): e92880. doi:10.1371/journal.pone.0092880.
  3. ^ a b c d 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.
  4. ^ a b Smeets, R. M. M.; Keyser, U. F.; Krapf, D.; Wu, M.-Y.; Dekker, N. H.; Dekker, C. (2006). "Salt dependence of ion transport and DNA translocation through solid-state nanopores". Nano Letters 6 (1): 89-95. doi:10.1021/nl052107w.
  5. ^ Piovesan, A.; et al. (2019). "On the length, weight and GC content of the human genome". BMC Research Notes 12: 106. doi:10.1186/s13104-019-4137-z.
  6. ^ Rubinstein, M.; Colby, R. H. (2003). Polymer Physics. Oxford University Press. ISBN 978-0-19-852059-7.
  7. ^ 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.
  8. ^ Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. (1996). "Characterization of individual polynucleotide molecules using a membrane channel". Proceedings of the National Academy of Sciences 93 (24): 13770-13773. doi:10.1073/pnas.93.24.13770.
  9. ^ Briggs, K.; Kwok, H.; Tabard-Cossa, V. (2014). "Automated fabrication of 2-nm solid-state nanopores for nucleic acid analysis". Small 10 (10): 2077-2086. doi:10.1002/smll.201303602.
  10. ^ Manning, G. S. (1969). "Limiting laws and counterion condensation in polyelectrolyte solutions I. Colligative properties". The Journal of Chemical Physics 51 (3): 924-933. doi:10.1063/1.1672157.
  11. ^ 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.
  12. ^ "kmer_models: expected current levels for k-mers (legacy R9.4, 450 bases per second, 6-mer table)". GitHub. Oxford Nanopore Technologies. Retrieved October 25, 2026.
  13. ^ 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.
  14. ^ 2019 02 19 17 20 38 fields of dna molecules at 100x (Video). Epic Quest Bio. February 19, 2019.
  15. ^ a b "Company records". Molecular Reality Corporation. October 2026.
  16. ^ Schrödinger, E. (1944). What Is Life? The Physical Aspect of the Living Cell. Cambridge University Press.
  17. ^ Bell, N. A. W.; Keyser, U. F. (2016). "Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores". Nature Nanotechnology 11 (7): 645-651. doi:10.1038/nnano.2016.50.
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