Finding and characterizing the complexes of drug like molecules with quadruplex DNA: combined use of an enhanced hydroxyl radical cleavage protocol and NMR.

Structural information on the complexes of drug like molecules with quadruplex DNAs can aid the development of therapeutics and research tools that selectively target specific quadruplex DNAs. Screening can identify candidate molecules that require additional evaluation. An enhanced hydroxyl radical cleavage protocol is demonstrated that can efficiently provide structural information on the complexes of the candidate molecules with quadruplex DNA. NMR methods have been used to offer additional structural information about the complexes as well as validate the results of the hydroxyl radical approach. This multi-step protocol has been demonstrated on complexes of the chair type quadruplex formed by the thrombin binding aptamer, d(GGTTGGTGTGGTTGG). The hydroxyl radical results indicate that NSC 176319, Cain's quinolinium that was found by screening, exhibits selective binding to the two TT loops. The NMR results are consistent with selective disruption of the hydrogen bonding between T4 and T13 as well as unstacking of these residues from the bottom quartet. Thus, the combination of screening, hydroxyl radical footprinting and NMR can find new molecules that selectively bind to quadruplex DNAs as well as provide structural information about their complexes

Depiction of the changes in cleavage induced by the four drug like molecules.

<p>The changes in cleavage of the residues are depicted using the chair type quadruplex structure of the DNA with blue indicating protection and red indicating enhanced cleavage. The changes for the 5′ and 3′ tail samples are both shown. Darker colors indicate a larger percentage change in the extent of cleavage.</p

Effects of NSC 176319 on the intensities of the imino resonances of T4 and T13.

<p>The ratio of the integrated intensity of the T4 and T13 resonances to that of the G quartets is plotted as a function of NSC 176319 concentration. The region of the 600</p

Representative cleavage results in the presence and absence of a drug like molecule.

<p>The cleavage results for the hydroxyl radical cleavage of the sample with the dT₁₁ tail on the 5′ end in the presence and absence of NSC 91881 are shown along with the results of a DMS cleavage. The changes in cleavage due to the presence of NSC 91881 are depicted for each band in the histogram on the right.</p

The 600 MHz spectra of TBA in the presence and absence of NSC 176319.

<p>The samples were at 288 K with the TBA at 1 mM. The integrated intensity of the G quartet region is the same in each spectrum. The dashed lines are to indicate which resonances change position as a function of NSC 176319 concentration.</p