Home > Lab-blog > Battle for supremacy between G-Quadruplex DNA fluorescent probes

Battle for supremacy between G-Quadruplex DNA fluorescent probes

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.

  1. March 8, 2012 at 14:00

    Excellent update about the quest of fluorescent probes, I really enjoyed the article. In brief, this work is about the study fluorescent of fluorescent properties of 8-(2-Pyridyl)guanine (2PyG) in comparison with 2-Aminopurine (2AP) in DNA. We can learn how this internal fluorescent probe that mimic guanine can maintain the folding stability of GQ-DNA and posses a higher quantum yield in comparison to 2AP.

    If we analyze the structure of 2PyG we can see that the Watson–Crick and Hoogsteen hydrogen bonding will be not perturbed significantly after the addition of the pyridyl group in the 8 position in comparison to 2AP. In the research to obtain a more efficient quantum yield, more libraries of fluorescent groups in the 8 positions has to be studied. Not only for an efficient quantum yield, but to the search for a candidate that don’t disturb the resulting folding of DNA and an emission that don’t quench due to stacking or other arrangement in physiological conditions.

    About the presented results, If we compare with other GQ articles, we will note how structure and function are always correlated because these are two of the main properties that GQs posses but other systems lacks. In this case, I obtain good information about this probe, specially the CD spectrum of hTeloG9, K+ and the other fluorescent studies. But more structural details of this folding were absent or are material to continue this work. For example, it will be good to see how these 2PyG modifications affect the structure by presenting some molecular modeling or simulations and maybe some 2D HMR will help to the elucidation of these resulting structures. Also, this opens the door for some crystallographic studies or energy measurements of the resulting stability of these systems. And I say this because the melting experiments shows that there is a significant effect in terms of stability with these modifications with 2PyG and 2Ap.

    About the resulting approach of using a high emissive probe as a pre-requisite in the folding process, I have some doubts or questions…I know that sounds very logic, but folding processes always result in quenching of these type of signals? There’s no way that the probe under study can adopt a configuration or be in a place of the DNA that the quenching can be avoided? Some times, the answer is in greater simplicity instead of grater complexity.

    About the synopsis, It was well written, it could avoid some details of the introduction, but the description of the results is very clear. I’m sure that María will do an excellent talk due her expertise in the area. About the picture, is the best picture to describe this article, I like how internal fluorescent probes are described.

  2. Diana S.
    March 8, 2012 at 15:04

    Luedtke’s new addition to his study of the photophysical properties of the 2PyG analogue scores again, this time with the publication of another full article comparing G-substitution in oligomers of his 2PyG derivative to the 2AP probe, as well as T, used as control.
    Because the sequences the authors use are so well characterized, they have the advantage that simple CD spectroscopy provides sufficient evidence of the quadruplex formation and topology of the modified oligonucleotides. The rest of the data relies on absorption/emission spectra. Comparison between the possible folded states of the oligonucleotides shows that 1) 2PyG is relatively harmless to the folded states and 2) that 2PyG doesn’t suffer from the fluorescence quenching, so commonly observed in these examples. This, in itself is great, and something many of us would like to see in our own systems, but not enough for what they’re looking for. Sadly, the increase in fluorescence quantum yield alone is not distinct enough to provide direct evidence of the folded/unfolded state of the oligo. However, through the calculation of energy-transfer efficiencies, they manage to get a larger signal, which they propose as a probe for the folding of oligonucleotides to GQs. Although I only recall seeing this type of data processing from his own previous publications, and I’m unsure how common it may be (however, I’m ready to admit that I’m not a great expert in fluorescence spectroscopy), it’s nice that they can get around the initial “problem” of low sensitivity by further processing their data.
    I really like Luedtke’s work, and I’m happy to see the development of his research since we first took note of him in 2010. I believe this is the fifth paper he publishes using 2PyG since then… I find the paper very readable, and the figures are attractive; not flashy, but clear and relevant.
    Maria’s synopsis is a bit long for my taste, considering that it’s not too hard to explain the general idea of the paper. But it is thorough and I like her observation that 2AP wasn’t perhaps the best choice as G analogue in the context of GQs because of the lost interactions. The figure is very nice, flashy and relevant.

  3. AJS
    March 8, 2012 at 17:43

    This weeks other paper is from the Ludke group: the next in their series of publications on 8-(2-Pyridyl)guanine (2PyG) as a fluorescent guanine analogue to probe DNA structure. In this article, they compare 2PyG to a fluorescent guanine ‘analogue’, 2-aminopurine (2AP), with particular attention to their suitability to study quadruplex structure. They prepare DNA oligomers substituted with both in various positions, and study the resulting structures (CD spectra and melting experiments) and photophysical response of the probes, and energy transfer between them and unmodified bases (UV/Vis, emission, and excitation spectra). They also show that an initial assessment of photophysical properties can be made without the hassle of DNA synthesis, by titrating with GMP (guanosine monophosphate).

    Broadly they find that, while not a perfect analogue, 2PyG doesn’t disturb quadruplex formation in most cases, whereas this is not the case for 2AP; this is unsurprising given that 2AP is unable to form the signature quadruplex H-bonding pattern. Furthermore, they find that 2PyG is brighter, and more able to partake in energy transfer interactions with other bases; that is, in most cases it is a superior probe.

    The paper is clear and interesting, although a little long-winded compared to this week’s other paper (Chem. Commun., 2011, 47, 5437-5439). I am perhaps most interested by the titration with GMP, which is a really simple and neat means to assess the viability of this kind of probe. I haven’t see that experiment anywhere else, so I’ll be interested to see if anyone else had.

    Maria’s piece is detailed and interesting, covering not just this article but a lot of related work (customary in our group meeting presentations, but not always here); since I had already read the article I see this as positive, but I can understand why others might not. Either way, I look forward to hearing her expand her ideas on Friday.

  4. Berti
    March 8, 2012 at 19:33

    Ludke and coworkers presents new as well as previously published data on G mutations made to hTelo and cKit where a single G is changed by either: 8-(2-Pyridyl)-2’-deoxyguanine (2PyG), by 2-Aminopurine (2AP) or by Thymine (T) (in some instances). The stability, the morphology as well as the fluorescent properties are studied. 2PyG presents an increase in fluorescent quantum yield when incorporated into the DNA, the higher quantum yield is obtained when mutant DNA is in the GQ conformation as shown by CD. The changes in the GQ global structure and stability of the hTelo and the cKit sequences are reasonable for this type of mismatch. The same is true for the lost of stability. What is really interesting is the different impact of each different G to the overall stability of the GQ.
    Another interesting result that should be taken into consideration is the enhance fluorescent emission of 2PyG after its incorporation into the DNA. If enhancement is a normal trend for this type of C8 modified derivative it means that a brighter G analogue will make a better probe.
    The titration experiment using GMP presents very interesting results. A continuous red shift in the excitation spectra with quenching below 400 mM GMP and enhanced above it. The red shift is explained by the effect of the base stacking but what is happening at the molecular level to have this strange quenching behavior? This question brings me to ref. 25, a very need and useful ref.
    The article is of relevance to our current research, it makes us refresh a few important concepts: Stern-Volmer plots, energy transfer efficiencies, quantum yield, fluorescent quenching and red-shift due to stacking interactions, and fluorescent probes among many other non fluorescent related terms.
    I still have my doubts regarding the energy transfer phenomena. This is established in 2010 (ref. 17) but now is when I realized that another possibility is very likely to happened. It is possible that 2PyG has an excitation band that overlap with the excitation band of the DNA and produces the emission at 415 nm. What they are observing is a simple enhanced emission due to optimal distance between 2PyG and the adjacent base and not a transfer from the rest of the DNA to the 2PyG. How can this process be investigate and described more accurately?
    María did an excellent work on the blog figure for her synopsis but the text of it was too long.
    Overall this is a very good paper, easy to read due to the good narrative and the well organized data. It’s an ideal article for a deepest discussion in our group meeting.

  5. Ana Victoria
    March 8, 2012 at 21:34

    Luedtke and coworkers are interested in developing energy transfer reactions between modified and unmodified nucleotides to be able to obtain information about nucleobase arrangements, dynamics and electronic properties. A fluorescent nucleobase analogue was used as an emissive energy acceptor, and the unmodified residues served as energy donors. The energy transfer efficiencies can be interpreted in terms of the folded states of the nucleic acids. Quenching because of the base-stacking interactions can affect the energy transfer efficiencies. 2PyG has a higher quantum yield when incorporated into folded oligonucleotides and it participates in highly efficient energy transfer reactions in G-quadruplexes. 2AP and 2PyG were compared in this study to see if these findings were unique of 2PyG. 2PyG exhibited greater fluorescence quantum yields when incorporated into nucleic acids. Because of this, 2PyG can be used to quantify energy transfer efficiencies, however 2AP is less sensitive to structurespecific trends in energy transfer. 2PyG exhibited an enhanced fluorescent emission after being incorporation in the DNA. The Stern-Volmer plot of 2PyG exhibited a downward curvature resulting in the recovery of florescence at higher GMP concentrations. There was a red shift in the excitation spectra with quenching at concentrations lower than 400 mM of GMP. The plot for 2AP presents the contrary, it has an upward curvature.

    I liked the introduction of the article and how they talk about their past work. The article deals with relevant concepts to our research such as quantum yields, energy transfer, fluorescence, etc. Their experiments correlate well with their findings and it was nice to read. Maria’s synopsis was really good, she did a good job explaining the relevance and the experiments, however it was a bit long. The figure does a good job representing the fluorescent probes.

  6. Mariana
    March 8, 2012 at 22:17

    Guanine Quadruplexes (GQs) is a very interesting topic and even more interesting when trying to develop fluorescent probes for their detection. It is a plus to read about 8-aryl/(heteroaryl)-substituted 2’-deoxyguanosine derivatives, since our group is a pioneer in that regard. The cleaver part of the work done is the incorporation of these derivatives as part of G-rich oligo sequences capable of forming Oligo-GQs, without disrupting the main interactions (H-bonds, electrostatic interactions). In specific Dumas and Luedtke have started a history in the topic since they first reported the synthesis of 2-PyG a 2’-deoxyguanine derivative containing a 2-Pyridil group attached to the 8-position of the guanine base. The work reported in a JACS 2010 was characterized not only by the synthetic modifications, but for the particular increase enhancement in fluorescence presented by the 2-PyG after the self-assembly of the oligo sequences into GQs. They attributed the phenomenon to the guanine cation coordination-dependency that can enhance the photo excited lifetime and can increase the quantum yield of the guanine nucleotides causing an overall favorable energy transfer to the substituted residue. Curious for me was that although their main claim was the cation effect coordination, they didn’t explain the tendencies observed by using different monovalent cations, some how this was not under the scope of their discussion (cations such as Rb+ and NH4+, which presented the lower Tm’s, showed a higher energy transfer when compare to K+ or Na+). Almost a year later (2011) they presented a more comprehensive study by adding other substitution (at the 8 position) and studying the phenomenon to establish if the enhancement in fluorescence was associated to the metal cation coordination or if structural changes can modulate the effect. Once more the 2-PyG resulted to be the most prominent derivative to be use as a fluorescent probe to study GQ folding.
    Now they are presenting a comparison with a fluorescent probe commonly used, 2-aminopurene (2AP). They chose 2AP because they want to test it as a guanine mimic within a G-tetrad and compare it behavior with 2-PyG as fluorescent probe. I just have a doubt and it is why after mentioning in 2010 that 2AP “is a poor guanine mimic and not very bright due to its low molar extinction coefficient (_305 nm ) 5600 cm-1 M-1) and low quantum yield (Φ ≈ 0.005) when base stacked with guanine residues” they want to use it to compare with their system?
    I believe they new 2AP will be a really bad fluorescent probe and that it will even cause a destabilization to the tested GQ sequences. The double stranded results again favors the use of 2-PyG, but I guess most of the results were previously published. From my point of view this results should be incorporated in previous articles to highlight the greatness of the 2-PyG as a new fluorescent probe for GQ detection or other possible applications. I do like the article; it is like a review of their work incorporating a known derivative (2AP) and a control (T) to compare. I guess it completes their 2-PyG stories…
    Maria’s synopsis was long but well organized. I know she likes to give details to avoid misunderstanding. I will like to know what she thinks about my comment and maybe she has a hypothesis related to the different publications since I know she recurrently follows his work. About the picture I like it, very representative of the work done.

  7. Loruhama
    March 9, 2012 at 00:32

    In this article, Luedtke and coworkers present their studies of the photophysical characteristic of the guanine mimic 2PyG. They also studied the energy transfer between the modified nucleobases possessing an internal probe, and unmodified nucleobases. In these studies 2PyG served as an energy acceptor, while the rest of the unmodified nucleobases served as energy donors. When they incorporated the 2PyG into GQ, they revealed a highly efficient energy transfer reactions with these molecules. However, duplexes containing 2PyG exhibited very little energy transfer, but about the same quantum yield as 2PyG alone. They also compared 2PyG to AP, and they showed that 2PyG exhibited higher fluorescence and quantum yield than AP when incorporated into duplex DNA. They also specify that 2AP does not support the formation of stacked G-tetrads in intramolecular GQ, making 2AP a poor probe to quantify energy transfers.
    I really liked this article, and the introduction was very engaging, and they explain their data very well. Overall I think that they did a very good work, and the fact that this article is so relevant to our research makes it more interesting. Maria’s synopsys was good, although a little too long, but nevertheless she summarizes all the main parts of the article. Overall, I’m really looking forward to see this presentation. The picuture was very relevant too.

  8. Y. Melendez
    March 9, 2012 at 01:28

    Luedtke and coworkers compare the fluorescent properties of 8-(2-Pyridyl) guanine (2PyG) with those of 2-Aminopurine (2AP) in DNA. The developed energy transfer reactions between modified and unmodified nucleotides in order to extract information about the arrangements in the nucleobases, and their dynamic and electronic properties. Using 2PyG as an energy acceptor and the rest of the nucleobases, which aren’t modified, were used as donors. When 2PyG was incorporated into the GQ, it showed highly efficient energy transfer, but they showed didn’t show a great deal of energy transfer in duplexes. 2PyG also showed a higher quantum yield than AP in duplex DNA. The fact that 2PyG and other 8-substituted guanosines would be good as probes for energy transfer, because they stay highly emissive, even when stacking with other purine residues.

    I liked the article, I think the introduction really instructs us about the background of their investigation and the article as a whole was very complete. The fact that it is so related to our line of work makes it that much more interesting. As for Maria’s synopsis, I think it was pretty good, she might have gone too much into detail but it is very well written nonetheless. The picture was a pretty good representation of the article, so I liked it too.

  9. Rocio Roque
    March 9, 2012 at 01:48

    As the title readily states, this article is about the study of fluorescent properties of 8-(2-pyridyl) guanine (2PyG) as compared to 2-aminopurine (2AP) in DNA, and sometimes Thymine. DNA oligomers were prepared and substituted (small family of 8-substituted-2’-deoxyguanosine analogues) and their photophysical properties evaluated. It was seen that 2PyG exhibited an increase in fluorescent quantum yield once incorporated into DNA, it also participates in highly efficient energy transfer reactions in G-quadruplexes. 2PyG does not disrupt quadruplex formation and it is perhaps a better probe than 2AP.

    Maria’s picture was really nice and I think depicted the essence of the article. Her synopsis was also good and it explained all the relevant details and experiments that were carried out (though it was little bit long for me). But then again, I also thought the article was a bit long. Nevertheless, I thought it was a very interesting paper and I look forward to understanding those things that I still have doubts on, during Maria’s discussion tomorrow.

  10. Jean
    March 9, 2012 at 02:06

    A lot of information on DNA stability and structure can be acquired through FRET experiments. These experiments usually involve the replacement of wild type nucleobases with fluorescently labeled analogs. This article by Dumas and Luedtke explores the ability of 8-(2-pyridil)-2-deoxyguanosine (2PyG) to efficiently serve as an acceptor in a FRET mechanism, and thus give useful information. They also tested the analog’s effect on the stability of both; the GQ structures and duplex DNA. They found that 2PyG is not as destabilizing as other guanine analogs in both GQs and duplex DNA but it’s presence can induce changes in the type of GQ formed. In particular, 2PyG exhibited higher transfer efficiencies when the DNA was folded into a G-Quadruplex instead of being in duplex DNA. The difference in energy transfer efficiency of 2PyG is 50 higher than 2AP, the most commonly used reporter. This allows the differentiation between folded (GQ) and duplex DNA by using only fluorescence. This was previously prohibited by the low difference in transfer efficiency of other guanine analogues.

    I have to say that for an article of such scope, the narrative was very engaging. The authors explained the importance of key experiments in a very straightforward manner. The figures were good but not even necessary (they are always necessary of course) due to the good narrative.

    I like Maria’s picture, it has neon glow all over it and aims to expose some of the articles finer details in a pictorial manner. However the word internal should have been capitalized. The synopsis, while by no means bad, was too technical for the purpose of this blog. Someone with a general knowledge of chemistry would grasp some of it but I seriously doubt that it would be useful. I would have made it more general and left the discussion of previous work for the GM presentation.

  11. PGMS
    March 9, 2012 at 05:21

    The work done by Dumas and Luedtke is very appreciable as they could provide an alternative and highly sensitive means to differentiate DNA/RNA structures possessing variable base stacking configurations. As fluorescence phenomenon is a powerful technique to characterize the folding and enzymatic activities of biomolecules, development of internal fluorescent probes for direct readouts of local nucleobases arrangements, dynamics and electron transfer reactions is a great research of to-day as they highlight precisely a single nucleotide substitution.
    To compare the abilities of 2AP and 2PyG as guanine mimics,G-quadruplex sequences derived from hTelo and cKit were site-specifically modified at positions directly involved in G-tetrad formation. Three sites in hTelo (G9, G17, and G23) were selected for substitution with 2PyG, 2AP, or thymine (as a negative control). The global structure and stability of each oligonucleotide were assessed by using temperature-dependent circular dichroism (CD). In K+ containing solutions, the unmodified hTelo sequence (hTe-lowt) adopted a “(3+1) hybrid” structure that exhibited a CD spectrum with a positive peak at 288 nm, a shoulder at 270 nm, and a minimum at 242 nm. This same characteristic CD spectrum was observed upon replacing G9 with 2PyG, but resulted in significant thermal stabilization of the structure (DTm=+108C) due to syn conformational preferences shared by this position and 2PyG. This stabilization is consistent
    with the integration of 2PyG directly into the 5’-terminal G-tetrad of this natively folded G-quadruplex. The presence of 2PyG–G base-stacking interactions is also suggested by a 30 nm red shift in 2PyG’s excitation maximum (lmax 330 nm) upon its incorporation into hTeloG94.
    The results indicated that 2PyG is a very efficient fluorescent probe in with exhibiting enhanced fluorescence quantum yield (approx. 50-folds brighter) than 2AP. The compatibility and much brighter fluorescence of 2PyG makes to quantify precisely energy-transfer efficiences. Further, the stern-Volmer plots of 2PyG showed an unusual downward curvatures of F0/F while 2AP fluorescence, an upward curvature of F0/F upon titration of guanosine which indicated a recovery of fluorescence at high GMP concentrations. These results are consistent with the trends observed for 2PyG- and 2AP-containing oligonucleotides, suggesting the solutions with high GMP concentrations could mimic the high local nucleobase densities of folded nucleic acids.

    The work on Guanine mimics is awesome, I like the picture and the synopsis by Maria is very appreciable.

  12. Maxier A. S.
    March 9, 2012 at 08:50

    Luedtke et al present a energetic transfer reaction of a series of nucleobases for arrangement characterization. The FRET pair consisted of the modified nuecleobase (2PyG) would work as the acceptos and the unmodified the donor. The introduction of the 2PyG, high efficiency (4-10 folds) of the energy transfer was observed but also possible quenching due to stacking. For quantum yield studies, 2PyG showed higher yield in the folded oligonucleotide.
    In general I thought the paper was good, great narrative and discussion. The discussion was very good, didn’t left any wholes. They kept reminding the reader the usefulness of the system trough the paper.
    About Maria’s synopsis I think it was good, maybe she tried to cover too much, yet good summary of the paper. Her picture was really great.

  13. Marilyn
    March 9, 2012 at 09:45

    In this article, Luedtke and colleagues report their studies of the photophysical characteristic of guanine analogues 2-aminopurine (2AP) and 8-(2-pyridyl)-2’-deoxyguanosine (2PyG). To accomplish this they studied the energy transfer process between the modified nucleobases with the internal probe, and unmodified nucleobases. As they models for they use the human telomeric repeat (hTelo) and cKit promoter (cKit) and incorporated the 2PyG and 2AP in different positions, positions that are directly involved in the tetrad formation. Their studies revealed a highly efficient energy transfer reactions with these molecules, but in the other hand duplexes containing 2PyG exhibited very little energy transfer and about the same quantum yield as 2PyG alone. Also 2PyG when compared to AP showed a higher fluorescence and quantum yield than AP when incorporated into duplex DNA. CD experiments showed that 2AP does not support the formation of stacked G-tetrads in intramolecular positions, making 2AP a poor probe to quantify energy transfers. Stern–Volmer plots of 2AP and 2PyG fluorescence showed that upon titration with guanosine monophoshate (GMP), at higher concentrations of GMP can mimic the high local nucleobase densities of folded nucleic acids.
    The introduction of this article was very appealing, which makes you want to read more and that is always good. They data is well presented and explained. Overall for me the article was to long but the fact that they took the time to tide their experimental observations whit structural features of the assembly was very helpful for me, since I’m a very structure oriented person and this dealing has been my main problem when dealing with others fluoresces articles. Which takes my to the questions, haw does a head-to-head heteropolar stacking orientation affect the fluoresces? And haw is the polarity of the face determined? Haw does a syn or anti orientation affect the fluoresces?
    Maria’s synopsis was very helpful, but too long for my. The picture was very good too.

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