DNA interaction with small molecule results in turbidity due to aggregation at milli molar concentration. However, the same interaction at low micro molar concentration does not cause visible aggregation. Simultaneously, the low concentration complex solution shows good CD signature for DNA secondary structure. However, the higher concentration complex solution fails to show any DNA signature in the solution!
Please suggest an explanation for this strange observation. Also, is there a way I can stabilize the higher concentration interaction without causing aggregation. I have tried using acetonitrile thinking the small molecule to be hydrophobic in nature, this solved the aggregation problem but it abolishes the activity of the small molecule in question.
Please comment on this query.
Thank you in advance.
How very strange indeed! You mean you cannot detect any dsDNA from the CD signal?? Is the DNA still there?
Can I suggest firstly raising the salt concentration, which might reduce hydrophilic interactions. Though not really compatible with CD, you would know whether this aggregation is hydrophilic or not. Also you can try a SEC or native gel electrophoresis to confirm the molecular weight of this aggregation.
The possible cause for bad CD signal from higher concentration complex solution is probably the high turbidity of the sample. If you sample is turbid, try to reduce the turbidity by diluting your sample first and then use a thin wall quartz cell for measurement.
What is the nature of your small molecules, are they charged under the studied condition? If they carry opposite charge as compared to your DNA, then I would assume that the small molecules bind to DNAs, leading to charge neutralization of the complexes, and when both intra and inter-polymer interactions are present, you may end up with large aggregates. You could try to measure the zeta-potential of the DNA, small molecules and the complexes and see if this is the case~ On the other hand, if you have too many small molecules (when your DNA is in lower concentration), the excessive charge carried by the complex (on the same sign with your small molecules) would cause the complexes to repel each other, therefore, no aggregation. Again, you can check this out by measuring the zeta-potential of your samples.
One thing that you can try is to stabilize the higher concentration DNA without causing aggregation is to increase the concentration of the small molecules at the same time to avoid charge neutralisation~
First, find out whether the small molecule is actually soluble at the high concentration.
Thank you very much for your answers and my apologies for limited information on the small molecule. I have included a little further info below.
Further info: the small molecule is insoluble in water so stock solution was made in DMSO to a concentration of 40 mM. DNA is a short oligo of length 6 and 12 basepair.
I think your problem stems from the aqueous insolubility of the compound. At high concentrations, you are adding a lot of DMSO to the DNA solution, which is probably causing it to precipitate, along with the compound precipitating due to the water it is being exposed to.
@Adam B Shapiro, yes, this could be the issue. Is there any way I can overcome this problem without loosing its activity?
Compound aqueous solubility can sometimes be improved by changing the pH and/or reducing the ionic strength (salt).
I try to give some idea but I am not sure that my answer will fit for you or not.
Small molecules tend to bind or interact with DNA double strands. When small molecules introduced (with DNA) in micro gram or less the DNA keeps its double strand but if you increase further to mill grams, the DNA double strand become less stabilized (ionic, electrostatic) and no more double helical structures exist, results the medium become turbid. maybe some ionic stability or other way of trials could help.
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