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Structural studies of naphthalene diimide ligands with telomeric G-Quadruplex DNA

March 15, 2012 13 comments

Structural Basis for Telomeric G-Quadruplex Targeting by Naphthalene Diimide Ligands

Gavin W. Collie, Rossella Promontorio, Sonja M. Hampel, Marialuisa Micco, Stephen Neidle*, and Gary N. Parkinson*

J. Am. Chem. Soc., 2012, 134 (5), 2723; DOI: 10.1021/ja2102423

A synopsis by Maxier Acosta

Previously Neidle had reported a series on naphthalene diimide (ND) oligo G-quadruplex (OGQ) ligands with side-chains (n) of 3-5 carbons with N-methyl-piperazine end groups. They showed experimentally how it inhibited binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibited telomerase activity in MCF7 cells via the stabilization of OGQs (DOI: 10.1016/j.bmcl.2010.09.066). Now, in collaboration with Parkinson, they report the crystalline structure of each one of those naphthalene ligands with the addition of a two-carbons side-chain.

They first give an overview of the tendencies of the overall parallel OGQ (Gtel22) with each ND ligand. With the telomere sequence d(AGGG[TTAGGG]3) they highlight the stacking of two OGQs making a dimer interacting from the 5’ terminal G-quartet. But the ratio between the ND and each OGQ is 1:1. Taking this in consideration, when each ND is bound to the quadruplexes, they force the topology of the loops into parallel strands as first proposed in DOI: 10.1016/j.bmcl.2010.09.066. While going more into detail, stability studies via FRET and inhibition studies where done for each ND. In the case of the ND with a two-carbons side-chain, it didn’t enhanced by much the stability of the Gtel22 due to the inappropriate side-chain length to enable effective interactions (in the OGQ groove) between the protonated N-methyl-piperazine and the DNA backbone phosphates. Although the n=5 ND OGQ complex showed poor quality in its crystal diffraction, it was still higher than that corresponding to n=2. For the n=4 ND, the side-chains were too long to fit well into the grooves as indicated by the disorder of the chains leading to a decrease of strong specific contacts, yet it was still more stabilizing than n=5 ND. For n=3 ND, it was observed that the cation-phosphate interactions were specifically coordinated, making it the best ligand of the small library presented in the paper. The structural features for these ND ligands correlated well with the inhibition of two types of cancer cells (MCF7 and A549).

In the discussion they summarized the data in three major topics: (1) the 1:1 binding of ND and OGQs; (2) the importance of the electrostatic side-chain interaction with the groove; and (3) the retention of the parallel topology of the Gtel22. Also, as might be expected for scientists from a pharmacy school they maintain their focus on how biologically relevant these binders could be for anticancer treatments.

In general, I thought that this was a good OGQ-binder structural article. I know that our systems are difficult to crystallize, yet this type of studies can help us to understand them to a new level so we could also start talking about potential inhibitors among other things. In terms of the organization of the paper, I found confusing the fact that they do not address explicitly some of the figures. In the discussion it was not that clear for me why the NDs induced the parallel topology; so, for that I encourage you guys to read the reference that I mentioned at the beginning, which has additional useful experimental data that may help anyone in the same situation. Other than this, I wish I had seen all of the ND side-chains interactions with the groove (some of them are in the supplementary information).

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Raiders of the lost G-quadruplexes …in the human genome

March 14, 2012 15 comments

Small-molecule–induced DNA damage identifies alternative DNA structures in human genes

Raphaël Rodriguez, Kyle M Miller, Josep V Forment, Charles R Bradshaw, Mehran Nikan, Sébastien Britton, Tobias Oelschlaegel, Blerta Xhemalce, Shankar Balasubramanian* & Stephen P. Jackson*

Nature Chemical Biology 8, 301–310 (2012) doi: 10.1038/nchembio.780

A synopsis by Diana Silva Brenes

The authors of this week’s paper play detective to find out -with great detail- what exactly happens to a human cell when it’s treated with the versatile, potent GQ-binder, pyridostatin. Using a combination of biomolecular assays, the authors manage to give strong support for the in vivo formation of GQ-DNA in human cells, and show their role in the activity of the new drug.

Pyridostatin is shown to induce damage to cellular DNA, stumping their proliferation. This happens because cellular checkpoints, which revise DNA before continuing the cellular division cycle, detect the damage and signal to the cell that something is wrong. The cell stops in its tracks to try to correct the problem before it continues the cycle. The drug, however, isn’t too toxic and most cells can survive long-term exposition to it without undergoing apoptosis. Interestingly, inhibition of the checkpoints restores cell proliferation.

Many of the results rely on detecting the presence of γH2AX (a protein that indicates double strand breaks in DNA) as a way to follow damage done to DNA. In cells treated with pyridostatin, γH2AX is present during the DNA transcription and replication processes, pointing at damage to DNA occurring during both stages.

Next, the authors wanted to localize where in the DNA is pyridostatin taking effect. Fluorescence labeling of γH2AX and the telomeres (marked by the labeling of a telomere binding protein) didn’t show co-localization. It was, thus, necessary to modify the drug to add direct fluorescence labeling. Addition of an alkyne group to the drug allowed an in cellulo click reaction with an azide containing fluorescent dye. After making sure that the modified pyridostatin did not affect drug activity, staining of pyridostatin was performed and fluorescent spots (foci) were compared with the a fluorescently labeled human helicase reputed to bind and resolve GQ-DNA during replication. Good co-localization was observed, suggesting that pyridostatin was localized mostly at putative GQ-DNA sites. In another experiment they showed that addition of pyridostatin before of after “freezing” the cellular processes in formaldehyde gave almost identical results, suggesting that GQ structures are pre-folded even without addition of pyridostatin.

They then performed ChIP sequencing to try to figure out which genes (aka, DNA segment) were targeted by pyridostatin. They found several specific genes (mostly away from the telomeres) that sustained pyridostatin induced damage to DNA, and all of them had above average putative GQ sequences. However, not all areas enriched in putative GQ sequences were affected, suggesting that there are other important requirements for interaction.

A particularly affected gene was SRC as confirmed by checking for loss of its corresponding mRNA transcription activity. Out of 25 putative GQ sequences estimated for this gene, 23 of them could be observed to form QGs in vitro using CD and NMR spectra.

The effect of pyridostatin on the bioactivity of SRC was also evaluated. SRC is important for wound healing and motility of cells. Cells treated with pyridostatin displayed a reduced ability to heal. As a control, cells treated with another DNA-damaging drug (DOX), didn’t affect healing, proving that the deficiency was not due merely to DNA damage.

It was previously shown that pyridostatin binds to GQs with enough strength to resist polymerases. It is hypothesized that damage to DNA by pyridostatin is due to mechanical forces breaking the DNA during the cell’s attempt to transcribe or replicate DNA. The findings of this paper support the potential drugability of GQs in cells.

The data reported by this paper is really important for the field of GQ binders and raises large hopes for the future of the field. Being able to use GQ to recognize and regulate specific genes is a dream come true in drug design, and the authors present strong data as to the viability of this approach. As a chemist, it’s difficult to get used to the rather indirect type of evidence that supports these findings, making it hard for me to comment on this paper’s methods. However, the controls and the analyses they did appear to be adequate. Overall, I find the results in this paper to be really important to anyone in the GQ field.

Categories: Lab-blog, Uncategorized Tags: , ,

Battle for supremacy between G-Quadruplex DNA fluorescent probes

March 8, 2012 13 comments

Fluorescence properties of 8-(2-pyridyl)guanine “2PyG” as compared to 2-aminopurine in DNA

Anälle Dumas and Nathan W. Luedtke*

ChemBioChem 2011, 12, 2044–2051; DOI: 10.1002/cbic.201100214

A synopsis by María Del C. Rivera-Sánchez

The motivation of the work reported by Dumas and Luedtke is the development of internal probes for direct readouts of local nucleobases arrangements, dynamics and electronic properties (e.g., electron transfer reactions). Their strategy is based on the incorporation of internal fluorescent probes as energy acceptors in DNA, particularly in hTelo and cKit sequences that fold into oligo-G-quadruplexes (OGQs).

In this article the authors include many of their previously reported data related to 2PyG [Refs 17 and 18] in order to compare its properties with those of 2-aminopurine (2AP), a nucleoside that was not previously evaluated as an internal fluorescent probe for OGQs when directly incorporated into folded G-tetrads. Each publication has different pieces of the puzzle towards understanding the importance of 2PyG as a plausible fluorescent probe and how it compares with other potential probes like 2AP. Thus, from those “scattered” pieces of information the picture that emerges can be summarized in the synthesis of a small family of 8-substituted-2’-deoxyguanosine analogues (2PyG, 4PVG and STG) and the evaluation of their photophysical properties in CH3CN and H2O. The cool part is that the phosphoramidite versions of these analogues were synthesized and the nucleosides incorporated into strategic positions of hTelo and cKit OGQs. The impact on the global structure and stability of hTeloG9, hTelo17, hTeloG23, cKitG10 or cKit15 having 8-substituted analogues, 2AP or thymine directly incorporated into folded G-tetrads, was evaluated by means of circular dichroism (CD) and CD-melting assays. Experiments using the afore mentioned ss OGQs were done in K+-, Na+-, and Li+-buffer and were compared to data from ds hTeloG9, ds hTeloG17, ds hTeloG23, and ds cKitG15 in Na+-buffer. In addition, the proficiency of analogues like 2PyG, 2AP and thymine as internal fluorescence probes was assed by measuring the quantum yield (Φ) and energy-transfer efficiency (ηT) of the substituted-duplex and ss-OGQs.

The data gathered from these experiments points to 2PyG as an outstanding internal fluorescent probe due to its higher quantum yield (Φ), once incorporated into folded oligonucleotides (Φ = 0.03–0.15) versus the free nucleoside in water (Φ =0.02), when compared to all other nucleosides evaluated. In addition, when exciting at 260 nm, the energy-transfer efficiencies from unmodified bases to 2PyG are 4–10-fold higher in ss-OGQs than in the corresponding duplex DNA. This energy-transfer process is favored by the O6 ion coordination within the central channel of G-tetrads and is distinctive of GQ structures (not duplex DNA). When this phenomena is combined with the high molar absorptivity of DNA it results in fluorescence enhancements of 10–30-fold for 2PyG-containing OGQs versus the corresponding ss- or ds-DNA. This highlights the potential of using 2PyG as a fluorescent probe for the detection of OGQ formation at lower concentrations among other applications. Unfortunately, the Φ or ηT of 2AP-containing DNAs are much lower than those for 2PyG-containing DNAs.

The ideal internal fluorescent probe should have very little effect on the global structure of the system evaluated. Particularly, the effect of 2PyG incorporation within folded G-tetrads seems to be context dependent. For example, 2PyG have little impact on the global structure and positive stability of hTeloG9 in K+- or Na+-buffer do to the syn conformational preference shared by this position and 2PyG. However, even though G15 in cKit (wt) have an anti conformational preference, CD spectra suggest that the incorporation of 2PyG have little impact on the global structure, but caused a small decreased in the Tm of cKitG15. On the contrary, the incorporation of 2PyG at hTeloG23 (in K+ or Na+-buffer) just allows the formation of an OGQ structure where G23 is in a syn conformation that is mainly observed on Na+-buffer. As a general trend, considering all the data discuss in the article, we can say that base stacking and pairing interactions can sometimes overcome the energy barrier of a preferred glycosidic bond conformation stabilizing the resulting OGQ or ds-DNA structure. Still, 2PyG has to be strategically located within OGQs to minimize detrimental effects, although, similar substitutions with 2AP or thymine are much more significant. Regarding 2AP, a priori I would not consider it a good mimic of guanine when positioned directly into folded G-tetrads because it lacks a carbonyl at the C6 and the N1-H, which prevents the formation of at least three interactions essential for an effective participation in the formation of a G-tetrad. Therefore, I consider that the comparison of 2PyG against other 8-substitutted nucleobases as they did on ref. 18 is more appropriate than comparing it against 2AP. The system reported by Dumas and Luedtke might have applications on fundamental studies related to ODNs and/or OQGs dynamics and their electronic properties, but I don’t picture them into practical, biophysical or technological applications.

This was a nice article in which it the authors combined many previous results with new complementary data provides a better understanding of the true potential and limitations of 2PyG as an internal fluorescent probe. They also evaluated for the detrimental effect induced by 2AP when incorporated into folded G-tetrads. The experiments reported included the appropriate controls like those done using thymine-containing sequences. In addition, their experimental section includes appropriate details such as the preparation of the DNA samples used.

Energetics of cations moving within G-Quadruplex DNA

February 8, 2012 14 comments
Parisa Akhshi, Nicholas J. Mosey, Gang Wu*
Angew Chem Int Ed Engl. 2012 Jan 13. doi: 10.1002/anie.201107700.
PMID: 22241618
A synopsis by Mariana Martín-Hidalgo

Gang Wu and coworkers are presenting in this short communication a molecular dynamic simulation to elucidate the free-energy landscape for the movement of three monovalent cations (Na+, K+, NH4+)through the central channel of a tetramolecular oligo G-quadruplex (OGQ).  The importance of this work strikes in the potential use of OGQs as synthetic ion channels, an application that has been considered by a number of research groups in last decade.

To perform this study they divided the ion movement in two regions: inside the OGQ and at the entrance/exit region.  They found that for the inner OGQ region, Na+ showed a lower energy barrier (4-5 kcal/mol) when compared to K+ and NH4+ (13-15 kcal/mol).  They made reference to experimental data to backup these computational results, plus they argue that the ionic radii differences between them are responsible for the observed energy barriers.  K+ and NH4+ have similar ionic radii (1.51 and 1.66 Å for octahedral coordinate ions respectively) while Na+ (1.18 Å) being a little bit smaller can diffuse through the tetrads easily.

They also highlighted the possibility of having what they refer to as “leaks” through the tetrad (sideway movement of ions) instead of the proposed continuous ion movement through the central channel.  They basically found that the energy barrier for that ion movement is too high (50-60 kcal/mol) to make it possible, discarding this possibility. I have to bring an issue related to their expression in this paragraph (second page, first paragraph), because they mentioned and I cite: “ there has never been experimental proof that ions would not “leak” out from the side wall of the G-quadruplex channel”.  I know the authors are emphasizing in the study of ion movement for OGQ’s but we know that there are OGQs and supramolecular GQs (SGQs) self-assembled from a variety of guanine derivatives. So, in my view they should’ve specified that lateral ion movement haven’t been shown for OGQs (perhaps in a note), but it had for SGQs. Specifically, in 2006 the Davis group reported (JACS 128 (47), 15269) the first evidence of “sideway” displacement for cations in a four-tetrad (4T) SGQ (i.e. a hexadecamer formed from the dimerization of two octamers). The mechanism of exchange of course would be different in a 4T-SGQ like Davis’ when compared to Wu’s 4T-OGQ and we can discuss those on Fridays presentation.

In the second region, the entrance/exit sides, the energy barrier encountered for K+ and NH4+ was 20 kcal/mol, while for Na+ was 14 kcal/mol, both values correlate well with the experimental data reported in the literature. Another important point of discussion the hydration states of these cations.  The inner cations are fully dehydrated while the ones located at the entrance/exit positions might be hydrated because of the transition to/from the bulk media.

From my point of view the most important part of this report is their comparison with a potassium ion channel.  All the gathered computational data reveals that the use of this or related sequences as K+ channels need to overcome significant energy barriers, even for the smaller Na+, if they are to be used as synthetic ion channels.

Although my technical knowledge of how to perform MDS is minimal, they contribute a very important part for the development of a molecular research project.  I think it will be great to have some fundamental MDS studies for some of our derivatives in relation to the ion movement not only in organic media, but also in aqueous environments.  As we have discovered in recent years, not all SGQs behave the same even if they have the same central guanine core in common.  Cations play a very important role in controlling these assemblies and I believe there is a need to put more effort in this regard, in particular with our system.  Any volunteers?

Categories: Lab-blog Tags: , , ,

Moving some of our lab blog posts to GQW

February 1, 2012 2 comments

From this semester onwards, the article synopses related to G-quadruplexes, which used to be posted in the blog in our lab website will be posted here. This is partly in preparation from transitioning away from the .Mac service (where the website is still hosted) since it will stop working in June of this year. In addition, using this blog is likely to increase the exposure of our posts as evidenced by the previous post. I’ll be announcing other changes in our web related activities soon.

In the meantime, continue to follow the guidelines in preparing your article synopses and postings:

An appropriate synopsis and comment should contain the following elements (1-3 for the blogger and 1-4 for the commenter):

  1. A brief synopsis of the article’s central idea (rationale, finding). In this part of your comment you must address explicitly the following issues: (a) What is the significance of the article? (b) How novel are the results? (c) Is the length of the manuscript appropriate to its contents? (c) What would you have done differently regarding this issue?
  2. A critical assessment of the experimental methods & techniques. In this part of your comment you must address explicitly the following issues: (a) Did the authors performed appropriate control experiments? (b) Are the conclusions supported by the results? (c) Are the references appropriate and correct? (d) Whenever applicable, is the characterization provided for new compounds appropriate with regard to identity and purity? (e) What would you have done differently regarding this issue?
  3. A critical assessment of the narrative. In this part of your comment you must addressexplicitly the following issues: (a) Is the narrative engaging? (b) Was the problem put in the proper perspective? (c) Did the authors do a good job explaining the significance of their findings? (d) What would you have done differently regarding this issue?
  4. A constructive criticism of the blogger’s synopsis. In this part of your comment you must address explicitly the following issues: (a) Was the synopsis well written (spelling and grammar)? (b) Could a person with a general knowledge of chemistry be able to grasp the fundamentals of the article being discussed? (c) Did the blogger addressed the issues i-iii of these guidelines? (d) What do you think of the blogger’s pictorial representation of the article? (e) Did the blogger used supplementary material to aid in the understanding of his/her synopsis (e.g. hyperlinks)  (d) What advice can you give to the blogger to improve his/her future synopses?
Categories: blogging, Lab-blog Tags: ,

Structural elucidation of dimeric DNA G-quadruplexes

February 1, 2012 13 comments

Stacking of G-quadruplexes: NMR structure of a G-rich oligonucleotide with potential anti-HIV and anticancer activity

Ngoc Quang Do, Kah Wai Lim, Ming Hoon Teo, Brahim Heddi1 and Anh Tuan Phan

Nucleic Acids Research, 2011, Vol. 39, No. 21, 9448–9457, doi:10.1093/nar/gkr539

A synopsis by Marilyn García-Arriaga

In order to gain better understanding of the nature of this structures, Phan and colleagues reported the structural analysis of a dimeric OGQ with the sequence (GGGT)4 (T30695) in K+ solution. This dimer is composed of two identical propeller-type parallel-stranded OGQ subunits each containing three tetrads that are stacked via the 5’-5’ interface. NMR structural studies of the OGQ formed by T30695 and T40214 ((GGGC)4), in K+ solution, share similar 1D and 2D NOESY spectral features. Furthermore, preliminary CD studies show the positive band at 260 nm characteristic of a parallel-stranded OGQ, in contrast to a previous report. In order to perform a detailed structural analysis by NMR an accurate assignment of the signals is essential. This task is more challenging in systems of high symmetry, thus, to overcome this problem they prepared a T30695 analogue with a single guanine-to-inosine substitution, GIGT(GGGT)3 (). This modification greatly improves the NMR spectra of the assembly allowing the assignment of the signals without ambiguity. Not only does this derivative show the same structural characteristics in the 1D and 2D NOESY spectra, but it also show similar positive band in the CD spectra and pattern in the gel electrophoresis experiments. 15N-labeling of the guanine imino protons and other protons of J19 enabled establishing the correlations in the COSY, TOCSY HSQC and NOESY spectra. In contrast to what was previously reported, the moderate intensity of the intra-residue H8/H6-H10 NOEs suggest that all residues adopt anti glycosidic bond conformation. The combined evidence suggested that the resulting structure is a propeller-type parallel-stranded OGQ with a three-tetrad core and three double-chain-reversal loops.

Evidence of the 5’-end stacking to form the dimer was obtained from gel electrophoresis in which the migration rate of J19 was similar to that of 93del, an interlocked dimeric OGQ. In addition, the migration rate of J19 was slower than that of a monomeric propeller-type OGQ. Furthermore, solvent-exchange experiments reveal that the imino protons of guanines in the outer tetrads (5’-end) are protected from exchange with D2O. Also, additions of bases in the 5’-end of J19 disrupt the dimer formation. The solution structure of J19 was generated after distance-restrained molecular dynamics refinement in this structure the core of the quadruplex show a close packing across the interfaces of the tetrads. Also, the directionality of the hydrogen bonds was the same for each subunit and opposite between the two structures in the dimer. The thymine bases are projected outwards in a double-chain-reversal loop. In more details, the sugars from the two end-tetrads are contiguous to one another and the backbones of the two dimer subunits are aligned in a staggered mode, maximizing the overlap of the five and six membered rings on the interface. They were able to conclude this by the identification of NOEs correlations among the base and the sugar protons of the end tetrads.

Finally, in order to assess if those OGQs conserved anti-HIV activity, they performed a reverse ‘disintegration’ reaction assay using T30695, J19 and their derivatives. They concluded that the derivatives containing thymines at the 5’-end were less active, which can be attributed to the lost of their ability to form stacked dimeric structures.

Once again Phan and colleagues present an impressive amount of work, but most of all, an incredible level of analysis. At the experimental level, the work is well supported and all the proper control experiments were performed. In contrast, the narrative and presentation of the data was least successful, in my opinion, it lacks details in the arguments of some conclusions.