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September 2001
Vol. 4, No. 9, p 12.
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HTS for DNA binders

The development of small molecules that predictably bind to specific DNA sequences is important pharmaceutically. Such species have great therapeutic potential, especially as antitumor agents, through their ability to block the recognition of mutated sequences by proteins. But to develop an effective DNA-binding drug, a comprehensive picture of a compound’s interactions with base pair (bp) sequences must be attained.

Recently, Dale Boger and colleagues at the Scripps Research Institute (La Jolla, CA) introduced a simple, rapid, and nondestructive method for high-throughput DNA-binding analysis (J. Am. Chem. Soc. 2000, 122, 6382–6394) that improves upon common, less efficient techniques such as footprinting and affinity cleavage. The method involves the stoichiometric intercalation of fluorescent species into DNA hairpin structures, in which each well in a 96-well plate contains a hairpin with a unique bp sequence. When a DNA-binding agent is added to the plate, affinity and selectivity are measured by the subsequent decrease in fluorescence, which indicates the degree to which the intercalator has been displaced.

Netropsin

DNA hairpin

Recently, the scientists further developed this method and demonstrated its utility by applying it to a set of prototypical DNA-binding compounds (J. Am. Chem. Soc. 2001, 123, 5878–5891). By screening the known AT-rich sequence binders—distamycin A, netropsin, DAPI, Hoechst 33258, and berenil—against all possible combinations of 5-bp sequences, the researchers optimized important parameters, such as binder, hairpin, and intercalator concentrations. They also made highly parallel comparisons among the binding agents.

Not surprisingly, there was a high prevalence of affinity for AT-rich sequences by all of the molecules. But by statistically comparing the top sequence affinities for each, it was clear that netropsin was the most AT-selective, in that the 50 DNA hairpins that showed the highest netropsin affinity contained 100% of the 5-AT sequences and only 9% of the possible sequences with 2 or 3 ATs in a row. By comparison, distamycin A was the least AT-selective (88%/27%).

The researchers also carried out quantitative titrations by performing the assay at a range of binding molecule concentrations to calculate reliable binding constants, which are important for drug development.

With the parameters optimized, the assay can now be applied to more experimental small-molecule DNA-binding compounds. In addition, the Scripps scientists note that the nondestructive nature of the technique permits hairpin immobilization and the reuse of plates, which makes the screening of complete libraries of longer sequences economically feasible.

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