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PubMed · 12565895

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

Akiko Kusunoki·Naoko Miyano-Kurosaki·Hiroshi Takaku
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Abstract

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.

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Binding SitesDNA, Single-StrandedHIV Reverse TranscriptaseHumansNucleic Acid ConformationOligodeoxyribonucleotidesRNARNA, Transfer, LysRNA, ViralRNA-Directed DNA PolymeraseVirus ReplicationJournal ArticleResearch Support, Non-U.S. Gov't

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Akiko Kusunoki, Naoko Miyano-Kurosaki, Hiroshi Takaku. 2003-02-07. A novel single-stranded DNA enzyme expression system using HIV-1 reverse transcriptase.. https://doi.org/10.1016/s0006-291x(02)03067-x

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