LETTER TO THE EDITOR

Journal of Radioanalytical and Nuclear Chemistry, Vol. 218, No. 1 (1997) 93-95

A simplified method for the preparation of r deoxyribonucleoside triphosphates

F. Wu, Y. Wang, M. Yi Institute of Atomic Energy, P.O. Box 275(39). Beijing 102413, P.R. China (Received May 30, 1996)

A method for preparing cr deoxyribonucleoside triphosphates ([0~-32pIdNTP)* is described. By comparing the labeling procedure to that of WALSET8 et al. 1'2 the reaction of Nudease PI is saved in our experiments and only three steps are needed for a routine preparation, without decreasing the yield of [r which is over 92% based on 32pi. In this paper [0~-32pldATP is taken as an example to deseribe the labeling procedure, the suitability of our method has been discussed.

Introduction carrier-free orthophosphate in acid-free aqueous solution, 40 mCi/mmol. All other reagents and enzymes were from 32p-nucleotides have been widely utilized in the study of Sigma (USA). The labeling procedure was monitored with many areas of biological" interest3-5 and can be prepared a HPLC pump (Waters, USA), a 834 Chromato-processor conveniently by the method of WALSETrIet alJ ,2 including (Hitachi, Japan) and a NaI crystal radioactive detector [T-32p]nucleotides and [ot-32p]nucleotides. When for (China). [tx-32p]dNTP, there always needs a four-step procedure for a routine preparation:* Labeling procedures Step 1 32pi-->[~-32p]ATP Step 1: D'-32p]ATP, which acted as phosphate donor, was prepared according to the method of WALSETrIet al. 1 Step 2 [ct-32p]ATP + Y-dNMP x Step 2 [~/-32p]ATP+ 3"-dAMP • X polynucleotide kinase >ADP + [5'-32p]3'-dNDP

• . polynucleotide kinase ) [5"-32p]3"-dADP + ADP Step 3 [5'-32p]Y-dNDP NucleasePl )[32p]dNMP+Pi pH 9,0

Step 4 [32p]dNlV[P NMP kinase, NDP kinase >[Gt_32p]dNT P [5,.32p]y.dADP Y-phosphatase ~[32p]dAMP + Pi pH 6.0

This paper describes a simple modification to above This step was carried out as follows: the enzymes procedure with which Step 3 was omitted and only three included in Step 1 were inactivated by setting the reaction steps involved for preparating [ct-32p]dNTP, without vessel in water of 80 ~ for 8 minutes, then 0.2 volume of decreasing the yield of [~-32p]dNTP. In this method the solution (the volume of Step 1 was regarded as 1 volume) conversion of [5'-32p]3"-dNMP to [32p]dNMP was containing 4 mM 3'-dAMP, 15 units of polynucleotide completed just by an adjustment of pH, allowing the kinase was added, this reaction was allowed to proceed 3'-phosphatase, which exists in the same protein molecule 5-10 hours at 10 ~ After this, the pH of the system was with polynucleotide kinase,6 to show its maximum activity adjusted to 6.0 by 1M HAc, then incubating 30 minutes at and hydrolyze the 3'-phosphate of [5'-3gP]3'-dNDP 25 ~ (Fig. 2 depicts the progress of this reaction). If there quantitatively. still left a small amount of unreacted [T-a2P]ATP (e.g. > > 0. 5% of the total radioactivity), a treatment as follows Experimental was needed to deplete the remaining [~/-32p]ATP and avoid nonspecific labeling: adding 0.08 volume of 40 mM Chemicals and apparatus glucose, containing 2 units of hexokinase in it, to the KCI, MgCI2, HAc were obtained from commercial reaction vessel, incubating 30 minutes at 25 ~ sources and were of the highest grade produced in China. Step 3: This reaction was initiated by an addition of 0.8 32p was purchased from Amersham (England) as volume of solution, containing 50 mM Tris-HCl, 5 mM

* Abbreviations used: dNTP, deoxyribonucleoside triphosphates; DT'I', dithiothreitol; PEP, phosphoenolpyruvate; dATP, dGTP, dCTP, the deoxyfibonucleotides of adenosine, guanosine and cytosine, respectively.

0236-5731/97/USD 17.00 Elsevier Science B. V., Amsterdam 1997 Akad~miai Kiadt, Budapest Akad~miai Kiadt, Budapesl All rights reserved F. Wu el a].: PREPARATIONOF O~-32p-LABELEDDEOXIRIBONUCLEOSIDE TRIPHOSPHATES

DTT 4 mM PEP, 250 lag/ml pyruvate kinase and of reactions catalyzed by polynucleotide kinase and 100p.g/ml myokinase. This reaction was allowed to 3'-phosphatase that go simultaneously but at different proceed 30-40 minutes at 25 ~ speed under a specific condition, and thus causing some Purification of [(x-32p]dATP was carried out in a extent of complication. According to CA~n~ROr~et al.6 the DEAE-Sephadex A-25 column according to Scrmr~EL et activities of polynucleotide kinase and 3"-phosphatase are al. 7 two functions of the same protein molecule, each of which has its own optimum pH and other conditions for its Results and discussion maximum activity. Polynucleotide kinase shows a maximum activity in a broad pH range between 6.5 to 9.0, The method of WAr.SETHet al. is efficient and versatile meanwhile, the optimum pH for 3"-phosphatase is about for preparing many kinds of 32p-nucleotides. When for 6.0 and its activity is considerably restricted at pH 9.0. So, [a-32p]dNTP, it has been simplified further in our in our experiments the reaction of phosphorylation experiments by saving the use of Nuclease P1 and by catalyzed by polynucleotide kinase was carried out at making use of the activity of 3"-phosphatase which pH 9.0, so that the activity of 3'-phosphatase was limited involved in the polynucleotide kinase. HPLC analysis of and the hydrolysis of the substrate 3'-dNMP (e.g. the three-step procedure for preparing [(x-32p]dATP, with a 3'-dAMP) was reduced without hampering the yield of 94.7% based on 32pi is shown in Fig. 1. phosphorylation. After this, the pH of the reaction mixture Generally, Steps 1 and 3 could be carried out smoothly was adjusted to 6.0, at this pH, the activity of in our experiments. But in Step 2, there included two kinds 3'-phosphatase was high enough to hydrolyze all of the 3'-phosphate of [5'-32p]3'-dNDP (e.g. [5'-S2P]3'-dADP) in 30 minutes. Based on this, the reaction of Nuclease P] in [ 32p]dAMP WntsEVa's method was saved in our experiments and the [ _32p]ATP reactions catalyzed by polynucleotide kinase and [ et-32 P]dATP 3'-phosphatase were controlled to proceed successively, quantitatively resulting [32p]dNMP (e.g. [32p]dAMP). By this, the labeling procedure was simplified and the 32 Pi possibility of diluting the specific activity and decreasing 1. -~._.r~ /77 ~ the radiochemical purity of the products by the - ) contaminant of enzymes and nucleotides which exists in 3..,,-J'~, Nuclease P1 was ruled out. The time course of Step 2 in our min. experiments, indicating the reaction progress and the influence of pH, is shown in Fig. 2. Fig. l. The HPLC analysis for the three-step procedure in our We have also discovered that our method may be the experiment. Using 200 • 4.6 nun C18 column, eluted by 17.5% methanol, containing 50 mM KH2PO4, flow rate: 1 ml/min. All the only choice when preparing [(x-32p]dGTP, for the percentages of radioactivity were given out directly from the Nuclease Pz, which was routinely included in WALSET"a'S chromato-processor (Mode-834, Hitachi); Step 1: 32pi 4%, h'-32pIATP method, showed very little activity against the 96%, Step 2: 32Pi: 4.3%, [32 P]dAMP 94.8%, 32P-glucose 0.9%, Step 3: 3'-phosphate of [5"-s2P]3"-dGDP. When Nuclease P] was 32 32 32 Pi 4.4%, P-glucose0.9%, I0~- P]dATP94.7% used to hydrolyze the [5'-32p]3'-dGDP after Step 1, even after a incubation of 3-4 hours at 25 ~ there still remained more than 54% of the [5"-32p]3'-dGDP unhydrolyzed. We concluded that the 46% of the 100--< hydrolyzed product, [32p]dGMP, was resulted not by 1 Adjust pH to 6.07I Nuclease P~ but mainly by the 3'-phosphatase, for this 80. percentage was similar to that of [32p]dGMP when Nuclease P] wasn't added and only by prolonging the 60- incubation time of Step 1 in a constant time. Further- "6 [,-'=PIATP "t more, Nuclease P1 in our experiments often resulted an o 40. 2 3: [3=P]dAMP 3 ,~~ unknown radioactive contaminant, its peak appeared rv" between that of [32p]dGMP and [(x-32p]dGTP in the 20- HPLC spectrum. By the three-step procedure, we have successfully prepared [(x-32p]dATP, [ot-S2p]dGTP and [ot-s2p]dCTP, all 0 , llllllllll~llllllllll~lllllllllllllll,I I Ill1 I" with a yield of more than 92% with respect to 32pi. Only 1 2 3 4 5 6 7 8 9 when for {~-32p]dCTP, because the 3'-phosphatase showed Time, hours a much higher activity against the 3"-dCMP in Step 2, usually more than 70% of the 3'-dCMP was hydrolyzed Fig. 2. The time course of Step 2. The analytical condition is identical within 3 hours, even at pH 9.0. For this reason, we have to to that of Fig. 1

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