Reduction of gene expression by a hairpin-loop structured oligodeoxynucleotide: Alternative to siRNA and antisense

• Published in: Biochimica et biophysica acta Vol. 1790; no. 10; pp. 1170 - 1178
• Main Authors: Kwok, Terry, Heinrich, Jochen, Jung-Shiu, Jiunshan, Meier, Michelle G., Mathur, Srikanth, Moelling, Karin
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2009
• Subjects: Antisense; Base Sequence; Cell Line; Cell Line, Transformed; Flow Cytometry; Gene Expression Regulation, Viral; Gene Silencing; HIV Reverse Transcriptase - genetics; HIV Reverse Transcriptase - metabolism; HIV-1 - genetics; HIV-1 - physiology; Humans; Hydrolysis; Nucleic Acid Conformation; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - genetics; Oligodeoxyribonucleotides - metabolism; Oligonucleotides, Antisense - genetics; Poly U - genetics; Poly U - metabolism; Polypurine tract; Reverse Transcriptase Polymerase Chain Reaction; Reverse transcriptase-RNase H; Ribonuclease H - genetics; Ribonuclease H - metabolism; RNA, Small Interfering - genetics; RNA, Viral - genetics; RNA, Viral - metabolism; siRNA; PBS; RT; DTT; nt; Antisense; siRNA; FACS; EGFP; Reverse transcriptase-RNase H; Gene silencing; as; PPT; PMSF; RT-PCR; Polypurine tract; NP-40; ODN; HA; RNase H; GAPDH
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2009.05.017

We previously described the inhibition of HIV-1 replication by a 54-mer hairpin-loop structured oligodeoxynucleotide (ODN) A, which binds the polypurine tract (PPT) on HIV-1 RNA. ODN A was shown to lead to reduced viral RNA in virions or early during infection. Here we demonstrated that ODN A was able to cause hydrolysis of viral RNA not only by retroviral RT-associated RNase H but also cellular RNase H1 and RNase H2 in vitro. Furthermore, ODN A reduced gene expression in a dose-dependent manner in a cell-based reporter assay where a PPT sequence was inserted in the 5′ untranslated region of the reporter gene. The efficacy of ODN A was higher than that of its siRNA and antisense counterparts. By knocking down cellular RNases H, we showed that RNase H1 contributed to the gene silencing by ODN A but the possibility of a partial contribution of RNase H-independent mechanisms could not be ruled out. Our findings highlight the potential application of hairpin-loop structured ODNs for reduction of gene expression in mammalian cells and underscore the possibility of using ODN A to trigger the hydrolysis of HIV RNA in infected cells by cellular RNases H.