Ehricht R, Ellinger T, McCaskill J S
Department of Molecular Information Processing, Institute of Molecular Biotechnology, Jena, Germany.
Eur J Biochem. 1997 Jan 15;243(1-2):358-64. doi: 10.1111/j.1432-1033.1997.0358a.x.
In vitro amplification systems not only serve as a tool for the processing of DNA, but have also provided important model systems for the investigation of fundamental issues in evolutionary optimization. In this work we present a coupled amplification system based on the self-sustained sequence replication (3SR), also known as nucleic acid sequence-based amplification (NASBA), which allows the experimental investigation of evolving molecular cooperation. The 3SR reaction is an isothermal method of nucleic acid amplification and an alternative to PCR. A target nucleic acid sequence can be amplified exponentially in vitro using two enzymes: reverse transcriptase (RT) and a DNA-dependent RNA polymerase (RNAP). A system has been constructed in which amplification of two molecular species is cooperatively coupled. These species are single-stranded (ss)DNA templates (D1 and D2) of lengths 58 and 68 nucleotides, respectively. Coupling occurs when D1 and D2 anneal to each other via a complementary region (DB and DB') situated at the 3' end of each template. RT elongates the hybridized templates producing a double-stranded (ds)DNA of 106 base pairs (bp). This double strand contains two promoters, which are situated on either side of, and directly adjacent to DB, and which are oriented towards each other. These promoters specify two RNA transcripts encompassing, respectively, the D1 and D2 portion of the dsDNA. After hybridization of two primers (P1 and P2) to the transcripts (R1 and R2) and reverse transcription, the ss templates D1 and D2 are regenerated. Amplification cycles of D1 and D2 are coupled cooperatively via the common dsDNA intermediate. Under optimized batch conditions the system shows the expected growth phases: exponential, linear and saturation phase. The enzymes of the 3SR cycle tend to misincorporate nucleotides and to produce abortive products. In future experiments, we intend to use the system for studies of evolutionary processes in spatially distributed systems where new strategies for optimization at the molecular level are possible.
体外扩增系统不仅是一种处理DNA的工具，还为研究进化优化中的基本问题提供了重要的模型系统。在这项工作中，我们提出了一种基于自我维持序列复制（3SR）的耦合扩增系统，也称为基于核酸序列的扩增（NASBA），它允许对不断进化的分子合作进行实验研究。3SR反应是一种核酸等温扩增方法，是PCR的替代方法。使用两种酶：逆转录酶（RT）和依赖DNA的RNA聚合酶（RNAP），可以在体外对目标核酸序列进行指数扩增。构建了一个系统，其中两种分子物种的扩增是协同耦合的。这些物种分别是长度为58和68个核苷酸的单链（ss）DNA模板（D1和D2）。当D1和D2通过位于每个模板3'端的互补区域（DB和DB'）相互退火时，耦合发生。RT延伸杂交模板，产生106个碱基对（bp）的双链（ds）DNA。这条双链包含两个启动子，它们位于DB的两侧并直接相邻，并且彼此相对定向。这些启动子指定了两个RNA转录本，分别包含dsDNA的D1和D2部分。在两个引物（P1和P2）与转录本（R1和R2）杂交并逆转录后，ss模板D1和D2再生。D1和D2的扩增循环通过共同的dsDNA中间体协同耦合。在优化的批量条件下，该系统显示出预期的生长阶段：指数期、线性期和饱和期。3SR循环中的酶倾向于错误掺入核苷酸并产生无效产物。在未来的实验中，我们打算使用该系统研究空间分布系统中的进化过程，在这些系统中可能有分子水平优化的新策略。
