Download Minor Groove to Major Groove, an Unusual DNA Sequence

Yoshihiro Miura
CHE 341L
Critical Thinking Assignment
Minor Groove to Major Groove, an Unusual DNA Sequence-Dependent
Change in Bend Directionality by a Distamycin Dimer
Shuo Wang, Manoj Munde, Siming Wang, W. David Wilson
Background:
Drugs that target the minor groove of DNA to treat cancer, parasitic diseases, and bacterial infections
have recently garnered significant attention in the field of medical research. This article addressed
distamycin (Dst), one such polyamide capable of dimerizing and binding to various lengths of AT
sequences in DNA. The authors’ primary goal was to evaluate the stoichiometry, affinity, and
cooperativity of Dst to bind to various lengths of AT sequences of the minor groove in DNA, and to
discover the curving tendencies of the Dst – DNA complex. While the information contained in the
introduction of their paper did provide sufficient information to comprehend the rest of the article, this
would not have been possible without the reader having a basic understanding of DNA structure, as well
as a thorough understanding of lab techniques such as electrospray ionization mass spectrometry (ESIMS), surface plasmon resonance (SPR), ligation ladder assay and polyacrylamide gel electrophoresis
(PAGE). Personally, I was unfamiliar with SPR: a more recently developed technique that allows direct
detection of a molecule binding to a probe without the use of a molecular marker. This technique
utilizes a sensor chip that has one side coated with streptavidin, to which probes that may potentially
bind to another molecule are attached. Light is then reflected off the other side of the chip, into a
detector. If the molecules bind to the probes, there will be a measurable change in intensity of the
reflected light. Run in a time dependent manner, both the association constant (Ka) and dissociation
constant (Kd) between the molecule and the probe can be calculated. The ligation ladder assay was
another technique foreign to me, which involves using a ligating enzyme to conjoin pieces of DNA to
create longer, repeating sequences of DNA of varying lengths. This procedure, in combination with PAGE,
Yoshihiro Miura
CHE 341L
Critical Thinking Assignment
will yield bands in a single column where each band is a different multiple of the repeating DNA
sequence.
Methodology:
The three main methodologies used to obtain data concerning Dst-DNA binding were ESI-MS,
SPR, and PAGE. With ESI-MS, information concerning the stoichiometry and cooperativity of the Dst
binding to DNA could be gleaned. By comparing the heights and masses of each peak which represent
the different stoichiometric ratios between Dst and DNA, the most prevalent stoichiometries for the two
molecules can be discovered. Furthermore, the cooperativity of these molecules can be found by
comparing the peaks between the 1:1 compound to DNA concentration to the 2:1 to see which
complexes become more prevalent.
For surface plasmon resonance, by adhering various lengths of AT repetitions (ATAT, ATATA,
ATATAT) to the foil, in addition to reconfirming the stoichiometry and cooperativity of the two
molecules, the molecules’ affinities can be evaluated. Based on the number of DNA bound to the sensor
chip, the predicted response unit (RU) for monomeric binding was 30. Depending on how the
experimental RU deviates from this anticipated value, the binding stoichiometry established from ESIMS can be confirmed or denied. Furthermore, by plotting the RU versus the Cf value (the concentration
of free Dst not bound to the AT sequences) and observing the shapes of the slopes, the different
cooperativities between the AT repetitions and Dst can be reconfirmed. Finally, the affinity of the
molecules can be determined from this method by observing the two association constants, K1 and K2.
Using PAGE, by running the ligation ladders of various lengths at different Dst to DNA ratios, the
curvature caused in DNA by Dst binding can be observed by the retardation of migration in the gel.
Furthermore, by using ligation ladder assays on a length of DNA with two AAAAA sequences, two ATATA
sequences, and a mix of AAAAA and ATATA, then running them through a gel with Dst, the direction of
Yoshihiro Miura
CHE 341L
Critical Thinking Assignment
the curve caused by Dst binding can be compared to that of the natural curve caused by multiple
repeating adenine subunits in DNA.
Assumptions:
One assumption the researchers made was that Dst binds to duplex DNA similarly to hairpin
DNA. This assumption was made based on the ESI-MS data which showed similar dimeric Dst-DNA
binding patterns for duplex and hairpin DNA. In both cases, a 2:1 concentration ratio of Dst to DNA
yielded a significantly higher percentage of dimeric peaks. Using this data, the authors established the
validity of using hairpin DNA for their SPR studies. This assumption is within reason, being that if Dst
binds to hairpin DNA similarly to duplex, the hairpin DNA bound to the chip in SPR would mimic the
results of Dst binding to duplex DNA. While some may argue that because this experiment tests for Dst
binding to DNA in the minor groove – a characteristic only present in duplex DNA and not in a singlestranded hairpin, this assumption is invalid. However, the authors sequenced their DNA in such a way
that the hairpin would fold onto itself and essentially form duplex DNA and create a minor groove
containing the ATAT sequence. Therefore their initial assumption is cogent.
The researcher made another particularly important and logical assumption when interpreting
the results from the PAGE. When running the various ligated DNA sequences of AAAAA, ATATA, and
mixed AAAAA/ATATA, they saw most apparently in the AAAAA sequence, a retardation of band
movement. Previous research indicated that multiple repeating adenine residues cause a bending in
DNA, resulting in band hindrance. As the concentration of Dst was increased for the ATATA ligation
ladders, band movement was also impeded. The authors made a logical assumption that the hindrance
in the ATATA’s were also caused by DNA curving, despite not having absolute evidence curving occurred.
However, this assumption is logical, and most likely correct, because there are no other obvious factors
that could influence the bands to migrate at such a significantly restricted pace. While differences in
Yoshihiro Miura
CHE 341L
Critical Thinking Assignment
band movement is usually caused by the difference in size between the molecules within the bands,
owing to the ligation ladders, the exact size of the DNA fragments can be seen and compared to the
other lanes, ensuring this retardation is not due to a difference in molecular size, but much more likely,
shape.
Arguments:
When the authors conducted ESI-MS on their 1:1 and 2:1 Dst to DNA concentration samples,
they found the prevalence of the 2:1 Dst to DNA associated peaks increased in all three DNA sequences.
They argued this data implied that given a higher concentration of Dst, the polyamide will dimerize with
ATATA and ATATAT and have cooperative binding, whereas ATAT would dimerize to a lesser extent, and
have non-cooperative binding. In SPR, the authors argued that because the RU values of each DNA
sequence were double that of Netropsin, a known monomeric DNA binder, it supported their
stoichiometric data from ESI-MS. Additionally, when the RU values were plotted against the Cf values,
the hyperbolic ATAT line attested to non-cooperative dimerization whereas the sigmoidal ATATA and
ATATAT lines indicated cooperative dimerization, further supporting their conclusions from the MS.
From the slopes of the time dependent RU graphs, the K values of the Dst – DNA associations were
found, and it was also determined that a strong affinity between Dst and DNA existed. Finally, the data
acquired from PAGE, the authors argued, showed the dimerized Dst bent the ATATA and ATATAT
sequences causing the bands to migrate at a slower rate. ATAT on the other hand, due to its low second
binding constant, did not allow for the second Dst to remain associated with the monomerized DNA, and
therefore, no bending was detected. Their second gel produced data that compared the ligated AAAAA
sequences – which showed retarded band migration, to ligated ATATA sequences – which showed
similar hindrances. Furthermore, after contrasting these bands to the ligated mix of AAAAA/ATATA, the
retardation was minimized as Dst concentrations were increased. The authors thus argued that the
Yoshihiro Miura
CHE 341L
Critical Thinking Assignment
curve caused by AAAAA and Dst-ATATA bent the DNA in opposite directions, cancelling out the
hindrance in the mixed DNA sequence. After considering the bending angles, they also stated that
dimeric Dst binding to ATATA seemed to change the directionality of the curve away from the minor
groove towards the major groove.
Every single one of the above arguments presented by the authors were presented and
explained in a logical manner. While they do not provide alternative explanations for any of their data,
their reasoning is quite convincing, and it would be difficult to interpret their findings differently.
Furthermore, not only does each method have stringent controls, the resulting data obtained between
the three methods support each other and have no discrepancies between them that cannot be
explained. This article was successfully written in a clear and concise manner, and a non-expert in the
field would be capable of comprehending this research as long as they had a basic understanding of DNA,
and were familiar with the laboratory techniques employed in this experiment.
Conclusions:
The final points of this article are: distamycin binds to ATATA and ATATAT sequences in a
dimeric fashion with high affinity and positive cooperation, whereas it binds to ATAT both
monomerically and dimerically, but binds dimerically with lower affinity and non-cooperatively.
Furthermore, AAAAA and ATATA with dimeric Dst both cause DNA to curve, but each cause the
molecule to bend in opposite directions. Specifically, the latter causes the direction of the curve to shift
from the minor, towards the major groove. The authors were successful in accomplishing their main
goal: to evaluate the stoichiometry, affinity and cooperativity of Dst to bind to various lengths of ATAT
sequences in DNA and to discover the curving tendencies of the Dst : DNA complex. Their use of rigid
controls and overlapping experimental conclusions make their results quite definitive.
Yoshihiro Miura
CHE 341L
Critical Thinking Assignment
This article has systematically assessed how Dst binds to specific sequences of DNA in an effort
to provide more data so this molecule, or other similar molecules can be used for drug development.
Seemingly inconsequential information such as stoichiometry, affinity, cooperativity, or DNA curving can
be vital pieces of data, indespensible for the creation of new medication. The discovery that dimerized
Dst causes DNA to alter the bends from the minor to major groove can be applied to use the molecule as
an allosteric regulator, preventing transcription factors from binding to the major groove. Such
innovative ideas are crucial for scientific progress and understanding our environment. The authors
successfully extended our knowledge of DNA, and provided other researchers with a possible starting
point for new drug development. Future studies can be conducted to determine the exact molecular
structure of DNA when bound to Dst to provide a clearer picture of the interaction of the two molecules,
and further still, application of minor groove binding in treating specific illnesses.