Chemical enzymatic synthesis of (2′-5′)-oligoadenylates using nuclease from Spicaria violacea mycelial fungus

Abstract

2′-5′)-Oligoadenylates [ppp5′A(2′p5′A) n , 1] are oligomers of adenylic acid with (2′-5′)-phosphodiester bonds that are synthesized in cells of mammals from 5′-ATP in the presence of interferon and/or double-helix RNA. They are activators of latent endonuclease L, which hydrolyzes mRNA of viruses and is in several instances responsible for the antiviral effect of interferon [1][2][3]. Furthermore, (2′-5′)-oligoadenylates affect cell growth, differentiation, and proliferation and apoptosis [4,5], are active toward plant viruses [6,7], and induce cytokinic activity in plant tissues [8]. Various types of biological activity can appear not only for 1 but also for dephosphorylated derivatives [A(2′p5′A) n , 2] [9, 10].Existing chemical methods for synthesizing these compounds are complicated and multi-step. Methods for preparing (2′-5′)-oligoadenylates that include polymerization of AMP derivatives to form both (2′-5′)-and (3′-5′)-internucleotide bonds and subsequent treatment of the resulting oligomers with several highly purified enzymes that selectively hydrolyze (3′-5′)-internucleotide bonds and terminal phosphates have been described [11][12][13]. The drawbacks of these methods are that they are labor-intensive due to the need to separate and purify several individual enzymes and the duration of the enzymatic steps.Herein we report the ability to use the filtrate of culture liquid (CL) of the mycelial fungus Spicaria violacea to produce (2′-5′)-oligoadenylates. It contains phosphatase and nuclease, which can hydrolyze (3′-5′)-but not (2′-5′)-internucleotide phosphodiester bonds in the chemically synthesized polynucleotides with mixed (2′-5′)-(3′-5′)-internucleotide bonds.We used S. violacea BIM F-329 strain from the Belorussian collection of nonpathogenic microorganisms of the Institute of Microbiology of the National Academy of Sciences of Belarus. The conditions for cultivating the fungus and the preparation of the CL filtrate containing phosphatase and nuclease have been described by us [14].Mixed (2′-5′)-(3′-5′)-polyadenylates were synthesized by polymerization of 2′(3′)-AMP in dioxane using diphenylchlorophosphate and tributylamine [11,12].Enzymatic hydrolysis of oligo-and polynucleotides by the enzyme complex in CL filtrate from S. violacea was carried out in Tris-HCl buffer (50 mM, pH 6.0) containing MgCl 2 (10 mM) at 60°C.The compositions of mixtures produced by enzymatic hydrolysis of polyadenylates were analyzed by HPLC on a Waters chromatograph using a Nova-Pak C18 column (30 × 3.9 mm) with elution by CH 3 CN (7%) in aqueous KH 2 PO 4 (0.1 M, pH 4.38) for 30 min. The chromatographic mobility of individual compounds in the mixture was compared with those of oligoadenylates of known phosphodiester bonding and chain length that were prepared by the triester synthesis method [15].The presence in S. violacea CL of phosphatase and specific nuclease that can hydrolyze only natural (3′-5′)-phosphodiester bonds was proved by experiments on the action of CL filtrate on polyadenylic acid that contained only (3′-5...

Preparation of (2′-5′)-oligonucleotides based on 6-N-benzylaminopurineriboside using the Spicaria violacea fungal enzyme complex

“…Thus, nuclease from FCM of S. violacea performed selective hydrolysis of (3′-5′)-phosphodiester bonds in mixed (2′-5′)-(3′-5′)-oligomers 8 that contain nucleoside units of 6-N-benzyladenosine analogous to that which was observed for compounds based on adenosine [11]. The lack of reaction products with terminal 2′,3′-cyclophosphates indicates that treatment of oligomers 8 with the enzyme complex from FCM of S. violacea also caused hydrolysis of terminal 2′,3′-cyclophosphates in the oligomers.…”

“…We have previously shown that short dephosphorylated (2′-5′)-oligoadenylates (2) can be prepared by enzymatic hydrolysis of long-chain oligonucleotides containing mixed (2′-5′)-(3′-5′)-internucleotide bonds by using (3′-5′)-specific nuclease and phosphatase in the filtrate of culture medium (FCM) of the mycelial fungus Spicaria violacea [11]. The goal of the present work was to develop a chemico-enzymatic method for synthesizing (2′-5′)-oligonucleotides containing 6-N-benzyladenosine (3) moieties as the nucleoside units.…”