A novel single-stranded DNA enzyme expression system using HIV-1 reverse transcriptase.
In this study, we exploited a DNA enzyme expression system using the mechanism of HIV-1 reverse transcription in vitro. HIV-1 reverse transcription is initiated when its cognate primer tRNA (Lys-3) binds to the primer binding site (PBS) of the viral RNA template. Therefore, this RNA contains the HIV-1 PBS, the DNA enzyme, and a tRNA (Lys-3) at the 3(')-end of its RNA transcript, such that a single-stranded DNA (ssDNA) is synthesized by the HIV-1 reverse transcriptase. We constructed RNA expression vectors including the HIV-1 PBS, the DNA enzyme, and either a native tRNA (Lys-3) or one of two truncated tRNAs (Lys-3), Delta tRNA (Lys-3) and Delta Delta tRNA (Lys-3). The reactions of the pVAX1-Dz-tRNA (Lys-3), pVAX1-Dz-Delta tRNA (Lys-3), and pVAX1-Dz-Delta Delta tRNA (Lys-3) vectors with T7 RNA polymerase in vitro gave the corresponding RNAs. The liberated RNAs were treated with HIV-1 reverse transcriptase (HIV-1 RT) in vitro, which yielded the corresponding ssDNA. The cleavage assay results demonstrated that the expressed DNA enzyme has cleavage ability against the target sequence. Thus, we have found a new DNA enzyme oligonucleotide expression system using the HIV-1 reverse transcriptase in vitro.
Kusunoki
Akiko
A
Department of Industrial Chemistry, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Miyano-Kurosaki
Naoko
N
Takaku
Hiroshi
H
D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D006801:Humans; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D012313:RNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase