Unexpected Effect of an Anti-Human Immunodeficiency Virus Intermolecular Triplex-Forming Oligonucleotide in an In Vitro Transcription System Due to RNase H-Induced Cleavage of the RNA Transcript

Abstract

A 16-mer oligodeoxynucleotide (ODN) which specifically recognizes the polypurine tract (PPT) located upstream of the 3′ long terminal repeat (LTR) of human immunodeficiency virus (HIV) proviral DNA via triplex formation is shown to have a dramatic effect on in vitro transcription from the HIV-LTR promoter. In the presence of HeLa cell extracts, a shorter RNA transcript is obtained in the presence of the 16-mer ODN. This truncated RNA lacks about 200 nucleotides from its 3′ region. The PPT sequence is not responsible for this effect. Instead, this process involves a purine-rich sequence in the gag mRNA located around position +400. The imperfect hybrid formed between the 16-mer ODN and mRNA is precisely cleaved by RNase H contained in HeLa cell extracts. These data show that sophisticated control experiments must be designed before any conclusion can be drawn on the effect of oligonucleotides used in vitro and in cell cultures.