IMPACT OF MIXED-BACKBONE OLIGONUCLEOTIDES ON TARGET BINDING-AFFINITY AND TARGET CLEAVING SPECIFICITY AND SELECTIVITY BY ESCHERICHIA-COLI RNASE-H

All phosphorothioate mixed-backbone oligonucleotides (MBOs) composed o f deoxyribonucleotide and 2'-O-methylribonucleotide segments were stud ied for their target binding affinity, specificity, and RNase H activa tion properties. The 2'-O-methylribonucleotide segment, which does not activate RNase H, serves as a high affinity target-binding domain and the deoxyribonucleotide (DNA) segment, which binds to the target with a lower affinity than the former domain, serves as an RNase II-activa tion or target-cleaving domain. In order to understand the influence o f the size and position of the DNA segment of MBOs on RNase H-mediated cleavage of the RNA target, we designed and synthesized a series of 1 8-mer MBOs with the DNA segment varying from a stretch of two to eight deoxyribonucleotides in the middle, at the 5'-end, or at the 3'-end o f the MBOs. UV absorbance melting experiments of the duplexes of the M BOs with the complementary and singly mismatched RNA targets suggest t hat the target binding affinity of the MBOs increases as the number of 2'-O-methylribonucleotides increases, and that the binding specificit y is influenced by the size and position of the DNA segment. Analysis of RNase H assay results indicates that the minimum substrate cleavage site and cleavage efficiency of RNase H are influenced by the positio n of the DNA segment in the MBO sequence. RNA cleavage efficiency decr eases as the position of the DNA segment of the MBO.RNA heteroduplex i s changed from the 3'-end to the middle and to the 5'-end of the targe t strand. Studies with singly mismatched targets indicate that the RNa se II-dependent point mutation selectivity of the MBOs is affected by both the position and size of the DNA segment in the MBO sequence. (C) 1998 Elsevier Science Ltd. All rights reserved.