596 Bulletin o[ Experimental Biology and Medicine, N o- 6, 1999 BIOPHYSICSAND BIOCHEMISTRY In Vivo Oligonucleotide-Protein Interactions in the Blood P. P. Laktionov, A. V. Bryksin, E. Yu. Rykova, and V. V. Vlasov Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 654-657, June, 1999 Original article submitted September 16, 1998 The interaction of oligonucleotides with serum proteins was studied in vivo by the method of affinity modification. Alkylating oligonucleotide derivatives administered to animals induced the formation of oligonucleotide-protein complexes circulating in the blood for a long-time. Oligonucleotides in these complexes were partially protected from nucleases. The major oligonucleotide-binding proteins identified with specific antibodies were albumins and IgG. In the erythrocyte fraction no specific oligonucleotide-binding proteins were detected, while membrane-cytosolic leukocyte fraction contained an oligonucleotide-binding protein with a molecular weight of 72 kD. Key Words: oligonucleotides; affinity modification; serum proteins; immunoglobulins; albumins Oligonucleotides (ON) are successfully used for in- hibition of virus replication and regulation of onco- gene expression and can be used for creation of anti- viral and antitumor drugs [10]. The study of meta- bolism and interaction of ON with biopolymers in living organisms is important for evaluation of pos- sible adverse effects and improvement of their phar- macological properties. ON are distributed and re- distributed in organs and tissues through the blood and their lifespan depends on nucleotide-nucleotide bonds and the presence of modified terminal groups in their secondary structure [5]. Binding of ON with serum protein was demonstrated in vitro [2]. In the present study we investigated the inter- action of ON derivatives carrying 5'-terminal alky- lating group (4-[(N-2-chloroethyl-N-methyl)amino] benzylamine with serum proteins after intraperitoneal and peroral administration to BALB/c mice. MATERIALS AND METHODS Deoxyribooligonucleotides pTGACCCTCTTCCCATT m p(N)16 and pT(TC)6T -- p(N)l 4 were synthesized by phosphotriester method in solution [3]. The radio- active label 32p was introduced into ON by transferring Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Medical Sciences the terminal phosphate from ]t-32p-ATP (BIOSAN, specific activity 5• 3 Ci/mmol) to the 5' position of ON using T4 polynucleotide kinase (Sibenzim). ON were purified from reaction products by electropho- resis (20% polyacrylamide gel: 1/20bis-acrylamide, 8 M urea, 0.5 M EDTA, 50 mM Tris-borate buffer, pH 8.3), transferred to DE-52-cellulose (Whatman), and eluted from cellulose with 2 M LiCIO 4 in acetone (centrifugation at 14,000 rpm for 5 rain in an Ep- pendorf 5415 C centrifuge). The pellet was washed with acetone, dried, dissolved in H20, and the concen- tration of ON was determined spectrophotometrically. Molar coefficients were calculated as described pre- viously [8] on the basis of known e=260 for mono- and dinucleotides [9]. Specific radioactivity of syn- thesized ON was 25-100 Ci/mmol. Alkylating group was introduced by binding (4- [(N-2-chloroethyl-N-methyl)amino]benzylamine to 5'- terminal phosphate of ON [1]. The yield of ON deri- vatives and the presence of active chlorine in alkyla- ting group were determined by the reaction with 0.5 M sodium thiosulfate followed by analysis of reaction products by electrophoresis in 20% polyacrylamide gel (1/20 bis-acrylamide) containing 8 M urea, 1 M EDTA, and 50 mM Tris-borate buffer (pH 8.4). The yield of alkylating ON derivatives was no less than 90%. Modified ON were administered to BALB/c mice weighing 25-27 g in a dose of 0.5 mg/kg in 200 gl 0007-4888/99/0006-0596522.00 9 1999 KluwerAcademic/Plenum Publishers