A novel single-stranded DNA enzyme expression system using HIV-1 reverse transcriptase

• Published in: Biochemical and biophysical research communications 2003-02, Vol.301 (2), p.535-539
• Main Authors: Kusunoki, Akiko, Miyano-Kurosaki, Naoko, Takaku, Hiroshi
• Format: Article
• Language: English
• Subjects: Binding Sites; DNA enzyme; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; HIV Reverse Transcriptase - metabolism; HIV-1 reverse transcriptase; Humans; Nucleic Acid Conformation; Oligodeoxyribonucleotides - genetics; Oligodeoxyribonucleotides - metabolism; Primer binding site (PBS); RNA - metabolism; RNA, Transfer, Lys; RNA, Viral; RNA-Directed DNA Polymerase - metabolism; ssDNA expression vector; tRNA (Lys-3); Virus Replication - genetics; Virus Replication - physiology
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/S0006-291X(02)03067-X

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), ΔtRNA (Lys-3) and ΔΔtRNA (Lys-3). The reactions of the pVAX1-Dz-tRNA (Lys-3), pVAX1-Dz-ΔtRNA (Lys-3), and pVAX1-Dz-ΔΔtRNA (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.