CHLORIN-OLIGONUCLEOTIDE CONJUGATES - SYNTHESIS, PROPERTIES, AND RED LIGHT-INDUCED PHOTOCHEMICAL SEQUENCE-SPECIFIC DNA CLEAVAGE IN DUPLEXES AND TRIPLEXES

Conjugates of oligonucleotides with chlorin-type photosensitizers were prepared. Two chlorin moieties, CPP and CHEVP, characterized by a mod ified pyrrole unit bearing an aldehyde chain, were photochemically pre pared from protoporphyrin and heptaethylvinylporphyrin, respectively. These chlorin moieties were coupled through the carboxylic acid side-c hain (CPP) or aldehyde side-chain (CHEVP) to the 3'-activated phosphat e of oligodeoxy-nucleotides. Diamine or dihydrazide were used as linke rs. The resulting conjugates were purified by HPLC and characterized b y electrophoresis, UV-visible spectroscopy, and mass spectrometry. The photosensitizing properties of the conjugate of CHEVP with the 14-mer oligodeoxynucleotide TTCTTCTCCTTTCT were investigated using three dif ferent targets. A single-stranded 25-mer containing the complementary sequence of the 14-mer formed a double helix with the chlorin-14-mer c onjugate. A 24 base-pair duplex and a 41-mer hairpin with 18 base pair s and a five nucleotide loop formed triple helices with the conjugate. In all cases, upon irradiation with visible light (428 or 658 nm), pi peridine-labile sites at guanine positions were produced. The reaction required oxygen and was inhibited to some extent by sodium azide. The cleavage sites were correlated with the chlorin position in both the duplex and tripler structures. In the 41-mer hairpin, the most reactiv e guanines were those located in the loop region. The quantum yield fo r cleavage of the hairpin structure was determined to be about 10(-3), independent of the excitation wavelength. This modest value is largel y compensated by the high absorption of the chlorin in the red, making the conjugate highly efficient even under low light fluence. No effec t was found with a noncomplementary chlorin-oligonucleotide conjugate. These results show that site-directed damages to nucleic acid structu res can be achieved using oligonucleotide-chlorin conjugates and red l ight irradiation.