Russian Journal of Bioorganic ChemistD', Vol. 26, No. 1/, 2000, pp. 758-764. Translated from Bioorganicheskaya Khim(va, Vol. 26, No. 11, 2000, pp. 844-8.51. Original RusMan Text Copyright © 2000 by Vorobje~;Pvshnaya, Pyshnyi, Repkova, Venyaminova, Zenkova, Ira^ova, Scalfi-Happ, Seliger, Bonora, Za~'to~'a. Cleavage of RNA in Hybrid Duplexes by the E. coil Ribonuclease H: II. Substrate Properties of Oligonucleotides Containing Nonnucleotide Linkers P. E. Vorobjev*, 2 I. A. Pyshnaya*, D. V. Pyshnyi*, M. N. Repkova*, A. G. Venyaminova*, M. A. Zenkova*, E. M. Ivanova*, C. Scalfi-Happ**, H. Seliger**, G. M. Bonora***, and V. F. Zarytova* *Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent'eva 8, Novosibirsk, 630090 Russia **Section of Polymers, University of Ulm, D-89069 Ulm, Germany ***Department of Chemical Sciences, University of Trieste, 1-42127 Trieste, Italy Received April 18, 2000; in finalform,June 16, 2000 Abstract--The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethyl- enediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA com- plexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid com- plexes of the bridged oligonucleotides proved to be substrates for the E. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2-1.4-fold. It is the com- position of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3'-ends of each hybrid duplex involving a bridged oligonucle- otide. The presence of an inverted 3'-3'-phosphodiester bond at the 3'-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity. Key words: antisense oligonucleotides, bridged oligonucleotides, nonnucleotide insert, ribonuclease H, phos- phodiester bond inversion, thermostability of complementary complexes INTRODUCTION The cleavage of mRNA in hybrid RNA/DNA duplexes by ribonucleases H is a key mechanism of the inhibition of gene expression by antisense oligonucle- otides [2]. This necessitates the study of the ability of these antisense oligonucleotides to stimulate the RNase H-induced hydrolysis of complementary RNA. Bridged oligonucleotides containing oligomethyl- enediol and oligoethylene glycol [3-7], oligoarginine [8, 9], dideoxyribose [10, 11], and other linkers [ 12, 13] were earlier proposed as tools for the study of interac- tions in short oligonucleotide duplexes and triplexes and as potential gene-directed compounds. However, the ability of hybrid complexes formed by bridged oli- godeoxyribonucleotides to activate RNase H has not been studied systematically; only a few publications evidence their substrate properties [4, 5, 9]. In this work, we studied the effect of the hexameth- ylenediol and hexaethylene glycol linkers on the hybridization properties of bridged oligonucleotides 1For the previousreport, see [ 1 ]. 2To whomcorrespondence should be addressed;phone: +7 (383) 239-6275; e-mail: vorobyev@niboch.nsc.ru. and on the RNase H-induced RNA hydrolysis of hybrid complexes with bridged oligonucleotides. RESULTS AND DISCUSSION The effect of the modifications of the internucle- otide phosphodiester bonds in bridged oligodeoxyribo- nucleotides on the ribonuclease H activation was stud- ied on the degradation of a 20-mer oligoribonucleotide (rM) [1] in complexes with the native oligodeoxyribo- nucleotide and with oligodeoxyribonucleotides con- taining nonnucleotide linkers (hexamethylenedioi and hexaethylene glycol) or a 3'-terminal inverted internu- cleotide bond. The influence of the number of nonnu- cleotide linkers in the oligonucleotide was analyzed with 20-mer oligoribonucleotides consisting of various oligonucleotide fragments joined by hexaethylene gly- col bridges (NloMNlo, N8MN4^^N8, and N5^ANs^ANs^^Ns), whereas the effect of the length of the nonnucleotide insert was studied with oligomers consisting of three fragments (octa-, tetra-, and octa- mers) joined by hexamethylenediol and hexaethylene glycol bridges or by di(hexaethylene glycol)phosphate groups (pNsAN4^Ns, N8^ANa^^N8, and Ns^A^N4^A^Ns, respectively (Scheme 1). 1068-1620/00/261 i-0758525.00 © 2000 MAIK"Nauka/lnterperiodica"