Department of Life and Environmental Science, Chiba Institute of Technology, Narashino-shi, Chiba, Japan.
Nucleic Acids Res. 2011 Jan;39(2):589-98. doi: 10.1093/nar/gkq794. Epub 2010 Sep 9.
DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tR(M)DtR(L), or one of two truncated tRNAs (Lys-3), tR(M)DΔARMtR(L) or tR(M)D3'-endtR(L). Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations.
DNA 酶比核酶更容易制备，对化学和酶的降解也不那么敏感；然而，尚未开发出 DNA 酶表达系统。在本研究中，我们利用 HIV-1 逆转录（RT）的机制在 DNA 酶表达系统中进行研究。我们构建了 HIV-1 RT 依赖性慢病毒 DNA 酶表达载体，包括 HIV-1 引物结合位点、DNA 酶以及天然 tRNA（Lys-3）、tR（M）DtR（L）或两种截断 tRNA（Lys-3）之一，tR（M）DΔARMtR（L）或 tR（M）D3'-endtR（L）。慢病毒载体介导的 DNA 酶表达在 SupT1 细胞中显示出对 HIV-1 复制的高度抑制作用。我们还在长期测定中证明了这种方法的有用性，发现 DNA 酶可防止 HIV 逃逸抑制。这些结果表明，HIV-1 RT 依赖性慢病毒载体衍生的 DNA 酶可防止逃逸突变的出现。
