I am using G-rich oligonucleotides to form corresponding G-quadruplex DNA using a standard protocol. Specifically, I am using 22mer HTelo and 22mer human oncogene promoter with Tm of 57.7 and 61.8 degree Celsius respectively. I am using UV-vis spectroscopy to determine Tm.
When I form the G-quadruplex their Tm drops ~ 5-6 degree Celsius to be 51.9 and 55.6 degree Celsius, respectively. My question if this is a common trend upon formation of G-quadruplex DNA or a change in Tm should not be expected ?
Another issue is that melting might in this case not be modelled as a pure dimeric thermal melting reaction. From Mergny you probably know that Tm not necessarily proportional to the chemical stability deltaG Article Analysis of Thermal Melting Curves
And from the article ".'Explicit analytic equations for multimolecular thermal melting curves" DOI: 10.1016/j.bpc.2015.04.001 you can see that the steepness around Tm depends also on the molecularity. The interpretation of stability depends on the baselines and the right mathematical model for the transition and finally a regression analysis. Maybe structure (un/folding) modeling is complex here. I suggest to study the literature of G Quadruplexes for raw data of thermal melting curves/plots (not only Tm) to study systematic trends. Finallly, I tend to agree with the answer of Dikeos Mario Soumpasis
Ι think you should expect that a smaller uv hyperchromic effect and therefore an apparent destabilization should be expected upon quadruplex formation but I would like to see the whole melting curves not just Tm in order to check this.
Thanks for your answer. Are there any references for this trend as I could not find one?
Unfortunately, the absorbance of UV of intact ordered nucleic acids versus that of their melted forms is not well enough understood except at the simplest level
which involeves base stacking and soliton like delocalization of the absorbed photons over the whole molecule.A satisfactory treatment requires quite a bit qvantum mechanics and is not feasible presently
Hi Mustafa. The stability of the system is dependent on ionic strength and of course potassium concentration for G4. What are the Tm experimental conditions for the expected value vs what you obtained? Which rate (K.mn-1) did you apply? Which buffer did you use? you can read papers from Jean-Louis Mergny for good G4-Tm practice.
Hi Laurent,
The papers from Jean-Louis Mergny were useful. I am aware of the effect of the experimental conditions and potassium concentration.
I only have Tm of the single stranded oligonucleotide as reported by the manufacturer, but I do not have a Tm value for the G4 I am forming to compare the results. My question was what trend is expected upon G4 formation, should it be an increase or decrease in Tm?
Thanks
Another issue is that melting might in this case not be modelled as a pure dimeric thermal melting reaction. From Mergny you probably know that Tm not necessarily proportional to the chemical stability deltaG Article Analysis of Thermal Melting Curves
And from the article ".'Explicit analytic equations for multimolecular thermal melting curves" DOI: 10.1016/j.bpc.2015.04.001 you can see that the steepness around Tm depends also on the molecularity. The interpretation of stability depends on the baselines and the right mathematical model for the transition and finally a regression analysis. Maybe structure (un/folding) modeling is complex here. I suggest to study the literature of G Quadruplexes for raw data of thermal melting curves/plots (not only Tm) to study systematic trends. Finallly, I tend to agree with the answer of Dikeos Mario Soumpasis
Hi Mustafa
well I think there is a confusion. The calculated Tm from the manufacturer should be the melting temperature of the double strand i assume? I mean, your oligo and its complementary strand. It is then not related to the Tm of your single strand structured oligonucleotide. For G quadruplexes, you should observe a decrease in absorbance measured at 295nm with increasing temperature.
Is it possible to have 29 deg C as melting temperature for a G quadruplex putative sequence?
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