SYNTHESIS AND CHARACTERIZATION OF OLIGODEOXYNUCLEOTIDES CONTAINING THIO ANALOGS OF (6-4)PYRIMIDINE-PYRIMIDINONE PHOTO-DIMERS

A method for the preparation of an oligodeoxynucleotide, 20 bases in l ength, containing centrally located thio analogues of (6-4) pyrimidine -pyrimidinone thymine photo-dimers is reported. The approach is based on the selective irradiation, at 350 nm, of a (TFT)-T-4S (T-4S = 4-thi othymidine) step within a 20-mer having the sequence: d(ACTCG-GACCT(T- 4S)CGCTGTGAT) . Conversion of the S-5-(6-4)/S-5-thietane pyrimidine-py rimidinone, initially formed, to its S-5-Dewar isomer is by a subseque nt irradiation at 300-nm. Both of the photo-dimer-containing oligonucl eotides were purified by HPLC (ion exchange and reverse phase) and cha racterised by base composition analysis. The S-5-(6-4)/S-5-thietane py rimidine-pyrimidinone containing 20-mer has a characteristic UV absorb ance at 320 nm and exhibits strong fluorescence when excited at this w avelength. As expected, conversion to the S-5-Dewar isomer abolished b oth the 320 nm absorbance and the fluorescence emission. The lengths o f the oligonucleotides produced allowed the formation of stable double -stranded DNA, by hybridisation to a complementary sequence. Examinati on of these duplexes by circular dichroism spectroscopy showed that th ey formed B-DNA, with Little changes to their gross structure as compa red to the parent duplex. However, local structural perturbations in t he region of the photo-dimer cannot be excluded. The S-5-(6-4)/S-5-thi etane photoproduct lowered the t(m) by 10.5 deg. C and the Dewar isome r by 12 deg. C. The degree of curvature induced in the DNA sequence by the introduction of the photo-dimers was assessed by analysing the mi gration of modified and unmodified multimer ladders on polyacrylamide gels. Both photoproducts induced considerable bending into the DNA. A comparison with a six-base-pair T tract, a bending standard that has a known bend angle of 19 degrees, gave values of around 47 degrees for the S-5-(6-4)/S-5-thietane product and about 28 degrees for the S-5-De war isomer. (C) 1998 Academic Press Limited.