Dissociation kinetics of actinomycin D from oligonucleotides with hairpin motifs.
The dissociation of 7-aminoactinomycin D (7-AM-ACTD) from d(ATGCATATGCAT), d(ATGCAT-T-ATGCAT), or d(ATGCAT-A-ATGCAT) at 20 degrees C cannot be adequately described by a single-exponential decay and requires a fit with two rate constants. The relative contributions of these two rate processes and their temperature dependence can be attributed to the coexistence of two conformational species in solutions. The slow dissociation rate corresponds to oligonucleotides in the dimeric duplex form, whereas the fast rate occurs with those in the hairpin conformation. The increased relative contribution of the faster component at higher temperatures is consistent with the more favorable thermal stability of the hairpin form. Studies with d(ATGCAT-TTT-ATGCAT) and d(ATGCAT-AAA-ATGCAT), which exist predominantly in the hairpin conformation, indicate that 7-AM-ACTD dissociates from these oligomers single-exponentially with rate constants comparable to or less than those obtained for the dimeric duplex of d(ATGCATATGCAT). Equilibrium binding titrations suggest that ACTD binds to hairpins as strongly as to the related dimeric duplexes, suggesting that the stacking geometry of the G.C base pairs at the dG-dC intercalating site of the hairpin stem is not greatly different from that of the dimeric duplex. The considerable variation in the dissociation rates of 7-AM-ACTD from hairpins, however, reflects the varying degrees of DNA minor-groove distortion of the stem duplex resulting from the hairpin loop formation and consequent interactions with the pentapeptide rings of ACTD. The plausibility of our interpretation is further supported by results from electrophoretic measurements, thermal melting profiles, and additional studies with hairpins containing a CGCG or GCGC stem.
Department of Chemistry, Tennessee State University, Nashville 37209-1561.
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Chen
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Jones
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Johnson
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry; D003609:Dactinomycin / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013997:Time Factors
D009838:Oligodeoxyribonucleotides; D003609:Dactinomycin; D012967:Sodium Dodecyl Sulfate; D004247:DNA