Shuo Wang, Manoj Munde, Siming Wang, W. David Wilson

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1 Background: Yoshihiro Miura 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 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 (ESI- MS), 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 (K a ) and dissociation constant (K d ) 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,

2 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 ESI- MS 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, K 1 and K 2. 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

3 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

4 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

5 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.

6 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.

20.320, notes for 9/13

20.320 Notes Page 1 20.320, notes for 9/13 Thursday, September 13, 2012 9:36 AM Coming Assignments 1st part of design project coming up, assigned 9/14 (tomorrow) and due 9/21. Last time We covered ITC