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...

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Bibliographic Details
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
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Summary: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.
ISSN:0006-291X
1090-2104
DOI:10.1016/S0006-291X(02)03067-X