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.