and the solution applied to a column (2.5 g, 10 mL) of Bio-Gel P-2(F j from Bio-Rad equilibrated with water. The crude product was purified by HPLC (Gen Pak-Fax column from Waters: A = 0.025 M Tris . HCI, pH 8.5; B = A + 1 M NaCI: linear gradient: 10 to 90% B over 45 min: flow rate = 0.7 mL min-' ; the chromatogram was monitored with a 2-channel diode array detector) and desalted on a bed of Bio-Gel P-2(Fj (2 g, water). A comparison of the UVjVlS spectra before and after desalting revealed no loss of product Spectrophotometric data were: oligonucleotide. eihO = 1.6 x 10, ~-' c m ~' : M n " ' p o r p h y r i n : ~, , , = 0 . 9 8 ~ lo5 ~-'cm-';hybridmolecule: calculated i,46h;~it,U = 0.62. observed A468/Aib4 = 0.63. Yield was 25% after final purilication and desalting (measured by UV spectroscopy at 264 nm). The HPLC retention time of the conjugate was 20.1 min compared to 22.6 min for the starting oligonucleotide. Each DNA cleavage reaction (see Fig. 1) was performed with 3.75 nM of the %labeled 35-mer (21 300 cpm nmol-; 9.6 nCi nmol-j. from 0.1 nM to 1 pM of Mn-trisMPyP 1Y-mer (DNA cleaver/target ratio between 0.026 and 260) and 400 p~ in nucleotides of double-stranded salmon testes DNA (3000 equivalents with respect to the 35-merj in a solution of 125 mM NaCl and 50 mM Tris . HCI buffer ( p H 8) Anncdling of the free Mn-trisMPyP 19-mer with the 35-mer was achieved by heating at 90 C for 3 min and followed by a cooling to 37 C within 4 h and overnight storage at 4Β°C. All assays (total volume = 16 pLj were performed at 4 C. For reactions without conjugate. the free 19-mer was hybridized with the 35-mer and then pre-incubated with 1 nM to 1 p~ of the parent DNA cleaver without vector Mn-TMPyP (pentoacetate of meso-tetrakis(4-N-melhylpyridinio)porphyrinatomanganese(irl): see ref.
[l I] for preparation) for 15 min at 4 C before addition of KHSO,. DNA cleavage reactions were initiated by addition of I mM KHSO, and maintained a t 4;C for 1 h. Reactions were then quenched by 40 mM Hepes buffer ( p H 8). and the mixtures heated at 90' C for 30 min (all concentrations listed are final concentrations). After cooling to 4 C. samples &ere diluted with 1 pL of yeast tRNA (IOmgmL-I) and 100 pL of 0.3 M sodium acetate (pH 5.2). precipitated with 300 pL ofethanol, and finally rinsed with 75 %ethanol and lyophylired. Fragments of DNA were analyzed by 20% polyacrylamidegel electrophoresis under denaturing conditions (7 M urea) as previously described"".