Andrew Jones

DNA Topology
Abstract:
DNA topology is an up and coming field of research investigating the supra and intermolecular
structures of DNA and related structures. A number of topological DNA structures have been
discovered and described. From its basic effects on gene expression to the more advanced nanomachinery it makes possible, understanding DNA topology is a worthwhile cause.
Introduction:
The relatively new field of DNA topology, the study of the supra and intermolecular structure
and confirmation of DNA and other cellular structures, has resulted in exciting findings, particularly
regarding nano technology and new biological engineering and lab techniques (1, 3, 5). Proteins are a
prime example of inter and supramolecular structure. Just as single amino acid proteins have their own
specific intermolecular structure, where they fold up on themselves, they can complex with other
proteins or molecules with either covalent or non-covalent interactions, forming a supra (alternatively,
super) molecular structure. In addition to new synthetic techniques, the research of topology has
resulted in an increased understanding of DNA structure and how the genome functions (1). Related
studies of DNA topoisomerases have coincided with these topology studies, granting further
understanding of the mechanisms (2, 7). Evidence suggests that DNA topology affects many proceses,
including transcription and gene regulation in a homeostatic fashion and DNA replication (4, 6). It's
important to understand the confirmations of these complexes.
A topological domain is a discrete section of DNA with the ends bound so that they cannot
rotate. The ends can be bound in a variety of ways: DNA can loop around in a circle, attaching to itself,
DNA can bind to the nuclear matrix, and it can also bind to the membrane. Circular DNA is most often
seen in bacteria. The entirety of the loops is seen as a topological domain. Chromosomal loops, which
are overall linear, have both ends bound to the nuclear matrix. In a similar fashion, some viruses can

have both ends bound to the interior of their capsid. The last example is a linear strand with both ends
bound to protein complexes (1).
A few variables exist that can be used to describe the topology of DNA in conjunction with
some equations (1). The linking number (Lk) of a double strand of DNA is determined by how many
times one strand crosses over an imaginary plane of which the boundary is the other strand (1). No
matter the confirmation of the double strand, the Lk is always the same, unless either or both strands
are broken and rotated (1). The second characteristic, twist (Tw) is the total amount of helical turns
under given conditions (1). Writhing (Wr) is the third characteristic, which is the shape the double helix
takes as a whole (1). This can be affected by changing Tw, as the helix compensates for the strain
produced by twisting or untwisting it, like a rope would (1). These values can be related to one another
by the equation Lk = Tw + Wr (1). As mentioned above, the topology of a double strand of DNA can
affect transcription and gene regulation. What affects this aspect more so than Lk, Tw, or Wr, is the
supercoiling of the DNA (4). Just as a rope loops around as it gets twisted tighter, DNA does the same.
As the number of base pairs per turn () goes down, the supercoiling goes up (1). Supercoiling is
defined quantitatively as a lowercase Tau (). In a relaxed configuration with no positive or negative
supercoiling, a DNA double helix follows this equation- Lk0 = Tw0 = N/ (1). However, real DNA
almost never follows this equation, so a different one must be used, which defines the linking
difference ()- = Lk Lk0 = Lk - N/ (1). When a strand is wound less than normal, is negative and
the strand is said to be negatively supercoiled. When a strand is wound more than normal, is positive
and the strand is said to be positively supercoiled (1). As expected, the torsional strain of supercoiling
is energetically unfavorable. It is energetically favorable for portions of negatively supercoiled DNA to
unwind so that the torsional stress is relieved (1). Methods besides merely unwinding exist in particular
cases to relieve torsional stress. If inverted repeated base pair sections exist, a cruciform can be created
(1). This happens when complementary strands unzip and then zip back together with themselves,
creating, as the name implies, a crucifix shape along the strand (1). Other confirmations exist, such as

Z-DNA, in which the strands unzip and zip back together in the opposite direction and H-DNA, in
which a three stranded portion is created, along with Hoogsteen triads, in which 3 bases hydrogen bond
together (1). A loop interlocked with another loops is referred to as a catanene (1).
Materials and Methods:
In October 2010, a paper was published by Dongran Han, Suchetan Pal, Yan Liu, and Hao Yan
entitled Folding and Cutting DNA into Reconfigurable Topological Nanostructures. Primarily, this
experiment dealt with using viral DNA to create a 100-nm Mbius strip, a loop-like structure with only
one side and only one edge. Additional techniques, similar to Japanese kirigami, known more
commonly as the folding and cutting method used to make paper snowflakes, can be used to create
topological structures. This involved the used of DNA oligonucleotides (referred to in the article and
here as oligos, for the sake of brevity), which are short sections of single-stranded DNA, to bind
longer single-stranded sections together. The Mbius strip was chosen for its ability to be easily
modified into different structures. Cutting a strip directly down the middle creates a loop twice as long
with two twists instead of one. If the strip is cut at about a third of the way in from the edge, two
interlocking strips are created. It creates a thinner Mbius strip interlocked with a longer strip with two
full twists. These results are repeatable, even with a DNA Mbius strip. The strip was constructed with
six single strands of viral DNA. Because the strip makes half a twist, it will end up being 11 strands
thick; the five on the outside rotate around the center one, eventually reconnecting to themselves. These
six single strands are connected around the length of the strip by sequential oligo strands, which zigzag back and forth from one side to the other. A total of 164 of these strands hold together the viral
DNA scaffolds. The Mbius strips were assembled by mixing scaffold and oligo strands in a 1:10 ratio.
The strands were at a 10 nM concentration in a tris-acetate-EDTA buffer which also contained 12.5
mM Mg2+. The mixture was cooled from 90C to 4C over a 10 hour period. Gel electrophoresis
followed by atomic force and transmission electron microscopy (ATM and TEM) was used to confirm
the formation of the intended product. Analysis via ATM and TEM confirmed correct topology. It was

discovered that the experiment yielded both left and right-handed chiral structures. The right-handed
product was favored because the DNA strands were right-handed and over-twisting would have
produced the right-handed product. The Mbius strip is interesting because the interlocking-loop
catenane structure can be created without having to cleave and ligate the strands as usual, using
topoisomerases, which first bind two strands of DNA, snip one, pass it over the other, and ligate the
snipped strand back together (2). The first product, which contains two full twists (and thus, is no
longer classified as a Mbius strip due to it having two sides and two edges) was produced by
removing the oligos attached to helix six. This was easily done, as the oligos attached to helix six had
both an exposed 3' and 5' tail with eight exposed nucleotides. When displacement strands were
introduced, they bound to the exposed ends and also pulled the unexposed bases off of the sixth helix,
separating it from both the fifth and the seventh. Though some of these kirigami strands showed an
open loop structure, most took the shape of a figure eight, forming a supercoil, easing the Tw strain.
The catanane double-ringed structure was formed by cutting the oligos between the fourth and fifth
strand, leaving the fifth and sixth as the smaller Mbius strip. Again, ATM and TEM were employed to
confirm the creation of the catanane.
Results and Discussion:
The yield of these structures (Mbius strips, double-twisted rings, and catanenes) were
determined to be 57, 66, and 74%, respectively. Keeping in mind the small amount of time that DNA
origami research unassisted by ligation has been around, these are quite promising numbers, which
only have room to increase as experimental methods improve. A number of interesting examples of
DNA-based nanomachines were mentioned, dating back as far as 2000. One example in particular
described a pair of tweezers formed from three strands of DNA and powered by DNA, producing
duplex bits of DNA as a waste product (8). Another, even more similar experiment by Ebbe S.
Andersen, was based around a box, made using the origami method, which had a controllable lid, and
could have many useful applications (9). Scaffolded DNA origami methods can also be used to

organize gold nano-particles with well controlled spacing and orientation, having possible applications
in a multitude of fields, including detectors and microscopy (10).
Conclusions:
Without being able to predict the future, the path these self-assembling DNA-based and even
DNA-fueled nanostructures is unknown. The application of DNA topology concepts and nanomachinery could be integrated with genetic research to work toward self-replicating, self-assembling
machines. Viruses capable of making the helices used in making the Mbius strips and other selfassembling DNA-based nano-structures could be made by modifying existing viruses. Nano-machine
potential aside, there is still knowledge to be acquired concerning the effects of topology on pathogenic
gene regulation and topoisomerases.

References:
1.

Mirkin, SM. DNA Topology: Fundamentals. Encyclopedia of Life Sciences. 2001

2.

Hardin, AH, et al. Direct measurement of DNA Bending by Type IIA Topoisomerases:
Implications For Non-equilibrium Topology Simplification. Nucleic Acids Research. (2011) first
published online March 17, 2011 doi:10.1093/nar/gkr109

3.

Mikelsen, MB, et al. Pressure-Driven DNA in Nanogroove Arrays: Complex Dynamics Leads
to Length- and Topology-Dependent Separation. Nano Letters. (2011) first published online
March 1, 2011 doi:10.1021/nl1044764

4.

Dorman, CJ and Corcoran, CP. Bacterial DNA Topology and Infectious Disease. Nucleic Acids
Research. (2008) first published online December 10, 2008. doi:10.1093/nar/dkn996

5.

Dongran, H, et al. Folding and Cutting DNA Into Reconfigurable Topological Nanostructures.
Nature Nanotechnology. (2010) first published online October 3, 2010.
doi:10.1038/nnano.2010.193

6.

Szambowska, A, et al. Coupling and Transcription and Replication Machineries in DNA

Replication Initiation: Evidence for Direct Interaction of E. coli RNA polymerase and the O
protein. Nucleic Acids Research. (2010) first published online September 9, 2010.
doi:10.1093/nar/gkq752
7.

Laponogov, I, et al. Structural Basis of Gate-DNA Breakage and Resealing by Type II

Topisomerases. PLoS ONE 5(6): e11338. doi:10.1371/journal.pone.0011338

8.

Yurke, B, et al. A DNA-fuelled Molecular Machine Made of DNA. Nature 406, 605-608 (10
August 2000) doi:10.1038/35020524

9.

Andersen, ES, et al. Self-assembly of a Nanoscale DNA Box With a Controllable Lid. Nature
459, 73-76 (7 May 2009) doi:10.1038/nature07971

10.

Ding, B, et al. Gold Nanoparticle Self-Similar Chain Structure Organized by DNA Origami.
Journal of the American Chemical Society, 2010, 132 (10), pp 3248-3249 first published online
February 17, 2010 doi: 10.1021/ja9101198