Enzvmatic coDolvmerization of nitroxide Jlabelled &ClLe derivativk with nucleoside diphosphates A. Hakam, I. E. Thomas, and A. M. Bobst Drprrfrwrl/ of’ C/wn~i.str~~, li~~iw~~si~~~ o/’ Cirwirlwrti. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG Sirfcirlrltrti. Ohio 45221, USA (Received 7 August 1979) Introduction Spin labelled nucleic acids are potentially excellent, sensitive tools for monitoring by e.s.r. the interaction of nucleic acids with a variety of biopolymers including proteins, other nucleic acids, and cell membranes’. Previously, spin labelled nucleic acids have been synthe- sized by limited chemical alkylation of hqmopolyribonuc- leotides. However, the chemical alkylation process does not allow one to obtain with certainty site-specific labelling with respect to the position of the base. Such specificity is desirable for a thorough theoretical analysis of the e.s.r. spectra of nitroxide labelled nucleic acid systems. Therefore, an alternative approach is necessary for the synthesis of high molecular weight spin labelled nucleic acids in which the position of the label is well defined. PNPase has been widely used for the synthesis of polynucleotides containing pyrimidine bases modified at the 5-position since the substrate specificity of the enzyme for various nucleoside 5’-disphosphate analogues is re- latively broad. The literature concerning the substrate specificity of PNPase is extensive; therefore the reader is referred to the excellent review of Godefroy-Colburn and Grunberg-Manago’. This paper contains a detailed ac- count for the synthesis via PNPase from Miuoumus lurrus of (RUGT, U),,, (RUGT, C), and (RUGT, A),,. In addition, the synthesis of the copolymer (IRUGT, U),, a nucleic acid lattice containing the biradical lRUGT, is described. A preliminary communication concerning the enzymatic incorporation of RUGT to form (RUGT, U),, has been reported3. Experimental Nucleoside-5’-diphosphates and poly(L-lysine) were obtained from Sigma Chemical Company. [4-(a- Abbreviations: PNPase. polynucleotldc phosphorylase: RUGT. (ribouracilglycosylamidotempo). N-[I-oxyl-2.2.6.6~tetramethyl-4- pipcridinyl]-O-[l-/~-~-ribofuranosyluracil-5-yl]-~lycolamide: IRLJGT. N(3) spin labelled RUGT: ppN. nucleoside diphosphate. 0141 8130~x0’010049~03$02.00 @ 1980 IPC Business Press Chloroacetamido)-2,2,6,6-tetramethylpiperidino-l-oxy] was purchased from Eastman Organic Chemicals. Polynucleotide phosphorylase isolated from zyxwvutsrqponmlkjihg Mic ~ro c m ~c w s Itrre w s was obtained from P-L Biochemicals, and (U),, fractions of various molecular weights were purchased from Miles Laboratories, Inc. The spin label was activated by reacting 4-(x- chloroacetamido)-2,2,6,6-tetramethyIpiperidino-l-oxy with a slight excess of an equimolar amount of sodium iodide in acetone. The reaction mixture was stirred overnight and centrifuged. The supernatant containing the activated spin label was added to a solution of 5- hydroxyuridine-5’-diphosphate synthesized according to Visser4. The alkylation was carried out in 0.5 M potassium phosphate buffer and the pH maintained at pH 9-10 with 1 N KOH. ppRUGT and pplRUGT were purified by paper chromatography (Whatman 3MM) eluting with ethanol:1 M ammonium acetate, pH 6.9, 7:3. The U.V. bands at R, =0.52 (ppRUGT) and Rf =0.7 (pplRUGT) were then eluted from the paper with water and lyophi- lized. The yield of both ppRUGT and pplRUGT was b 5 lo”,, with respect to the 5-hydroxyuridine-5’-diphosphate. The structure of RUGT and IRUGT elucidated by n.m.r. is reported elsewhere5. ppRUGT or pplRUGT was polymerized with nuc- leoside diphosphates done at various ratios of ppRUGT or pplRUGT to ppN. A typical incubation mixture (O.l- 0.11 ml) consisted of approximately 40 absorbance units of spin labelled and unlabelled nucleoside diphosphates and 1.5 units of polynucleotide phosphorylase in 0.1 M Tris-HCI buffer, pH 8.7, containing 0.8 /irnol MnCI, and 60. /l.g bovine serum albumin. After incubation for 6 h at 46 C the mixture was deproteinizedh with chloroform:isoamyl alcohol, 5:2. The nucleic acids were purified according to Bobst ’ by column chromatography on a Sephacryl S-200column (1.5 cm x 78 cm) eluting with 0.04 M ammonium bicarbonate. After dialysis successively Int. J. Biol. Macromol., 1980. Vol 2, February 49