A new optical strategy to determine the binding modes (intercalation vs groove binding) of small fluorescent organic molecules with calf thymus DNA was developed using two-photon absorption (TPA) spectroscopy. Two-photon excited emission was utilized to investigate a series of fluorescent nuclear dyes. The results show that TPA cross-sections are able to differentiate the fine details between the DNA binding modes. Groove binding molecules exhibit an enhanced TPA cross-section due to the DNA electric field induced enhancement of the transition dipole moment, while intercalative binding molecules exhibit a decrease in the TPA cross-section. Remarkably, the TPA cross-section of 4,6-bis(4-(4-methylpiperazin-1-yl)phenyl) pyrimidine is significantly enhanced (13.6-fold) upon binding with DNA. The sensitivity of our TPA methodology is compared to circular dichroism spectroscopy. TPA demonstrates superior sensitivity by more than an order of magnitude at low DNA concentrations. This methodology can be utilized to probe DNA interactions with other external molecules such as proteins, enzymes, and drugs.
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