Adenine-Thymine Base Pair Recognition by an Anthryl Probe from the DNA Minor Groove

Abstract Synthesis and DNA binding properties of geometric isomers, 9(3-aminopropyl)anthracene (APAC) and 9(N-ethyl)aminomethyl anthracene (N-Et-AMAC) are reported here. The aminopropyl side chain of APAC can position the amino function in the DNA grooves for hydrogen bonding with the heterocyclic bases. In contrast, the N-ethyl-aminomethyl side chain of N-Et-AMAC is too short to make contacts in the grooves. The DNA binding properties are examined using absorption, fluorescence, circular dichroism (CD), singlet–singlet energy transfer, and DNA melting experiments. In addition to calf thymus DNA (CTDNA), we examined poly(dA-dT), poly(dG-dC), poly(dA)-poly(dT), and poly(dI-dC). The spectroscopic and thermal studies indicate intercalation of the anthryl probes into the helix. The binding properties, however, depended on the side chain as well as the DNA sequence. The binding of APAC to poly(dA-dT) is preferred 3.4 times better over poly(dG-dC) while binding of N-Et-AMAC, the geometric isomer of APAC, is nearly the same with all the DNA polymers examined. Hydrogen bonding of the probe side chain from the minor groove of the helix is tested by using inosine-cytosine sequences. Consistent with the above data, APAC binds to poly(dI-dC) with binding constants similar to that of poly(dA-dT) sequences. Thus, both chemical and biochemical methods are used to test the hydrogen bonding of the probe side chains with the helix. The DNA sequence discrimination observed here clearly indicates the position of the amino function on the side chain plays an important role in DNA sequence recognition. The helix melting temperature of CTDNA is increased from 70°C to 80 and 82°C by APAC and N-Et-AMAC, respectively, indicating the helix stabilization upon intercalation of the probes. Intercalation is also supported by the CD spectra of the probe-DNA complexes. The probes are achiral and binding to the helix leads to induced CD spectra in the 300–400 nm region, away from the DNA absorption bands. The positive CD bands observed indicate intercalation of the anthryl chromophore with its long axis perpendicular to the base-pair dyad axis. Fluorescence polarization data clearly support intercalation of both probes into the above sequences, including poly(dI-dC), except that no polarization was observed with APAC bound to poly(dG-dC). This result is consistent with preferential binding of APAC to AT sites over GC sites and efficient quenching of anthryl emission at GC sites. Excitation into the DNA absorption bands results in sensitized emission from the anthryl probes and the efficiency of energy transfer depends upon the sequence. While AT sites sensitize anthryl emission efficiently, no sensitization was noted for GC sequences. These data clearly show that GC and IC sequences can be readily distinguished, in addition to AT sequences, by monitoring the sensitized emission from the anthryl probes. Thus, a comparison of the binding properties of APAC and N-Et-AMAC as well as a comparison of the binding properties of each probe as a function of DNA sequence clearly indicate the ability of the propyl side chain on the intercalator to position the hydrogen bonding group in the minor groove of the helix. Both probes can induce photochemical DNA strand damage that results in sugar-phosphate backbone cleavage. The photocleavage of the supercoiled DNA results in nicked circular DNA with little or no linear DNA suggesting that single stranded cleavage predominates. Most likely, the O2 of thymine in AT bases and O2 of cytosine in IC sequences interact with the aminopropyl side chain of APAC and this interaction provides the basis for the DNA nucleobase recognition by the small molecules.