Background Small RNAs are sequence-dependent negative regulators of gene expression involved in many relevant plant processes such as development, genome stability, or stress response. Functional characterization of sRNAs in plants typically relies on the modification of the steady state levels of these molecules. State-of-the-art strategies to reduce plant sRNA levels include molecular tools such as Target Mimics (MIMs or TMs), Short Tandem Target Mimic (STTMs), or molecular SPONGES (SPs). Construction of these tools routinely involve many different molecular biology techniques, steps, and reagents rendering such processes expensive, time consuming, and difficult to implement, particularly high-throughput approaches. Results We have developed a vector and a cloning strategy that significantly reduces the number of steps required for the generation of MIMs against any given small RNA (sRNA). Our pGREEN-based binary expression vector (pGREEN-DLM100) contains the IPS1 gene from A. thaliana bisected by a ccdB cassette that is itself flanked by restriction sites for a type IIS endonuclease. Using a single digestion plus a sticky-end ligation step, the ccdB cassette that functions as a negative (counter) selection system is replaced by a pair of 28 nt self-annealing primers that provide specificity against the selected target miRNA/siRNA. The method considerably reduces the number of steps and the time required to generate the construct, minimizes the errors derived from long-range PCRs, bypasses bottlenecks derived from subcloning steps, and eliminates the need for any additional cloning technics and reagents, overall saving time and reagents. Conclusions Our streamlined system guarantees a low cost, fast and efficient cloning process that it can be easily implemented into high-throughput strategies, since the same digested plasmid can be used for any given sRNA. We believe this method represents a significant technical improvement on state-of-the-art methods to facilitate the characterization of functional aspects of sRNA biology.

中文翻译：

---

方案：低成本快速高效地生成用于小 RNA 分析的分子工具。

背景 小 RNA 是基因表达的序列依赖性负调控因子，参与许多相关的植物过程，如发育、基因组稳定性或应激反应。植物中 sRNA 的功能表征通常依赖于这些分子的稳态水平的修饰。降低植物 sRNA 水平的最新策略包括分子工具，例如目标模拟物（MIM 或 TMs）、短串联目标模拟物（STTMs）或分子海绵（SPs）。这些工具的构建通常涉及许多不同的分子生物学技术、步骤和试剂，使得这些过程昂贵、耗时且难以实施，尤其是高通量方法。结果 我们开发了一种载体和一种克隆策略，可显着减少针对任何给定小 RNA (sRNA) 生成 MIM 所需的步骤数。我们基于 pGREEN 的二元表达载体 (pGREEN-DLM100) 包含来自拟南芥的 IPS1 基因，该基因被一个 ccdB 盒一分为二，该盒本身两侧是 IIS 型核酸内切酶的限制性位点。使用单次消化和粘性末端连接步骤，用作阴性（计数器）选择系统的 ccdB 盒被一对 28 nt 自退火引物取代，这些引物提供针对所选目标 miRNA/siRNA 的特异性。该方法大大减少了生成构建体所需的步骤数量和时间，最大限度地减少了来自远程 PCR 的错误，绕过了来自亚克隆步骤的瓶颈，并且无需任何额外的克隆技术和试剂，整体节省时间和试剂。结论 我们的流线型系统保证了低成本、快速和高效的克隆过程，它可以很容易地实施到高通量策略中，因为相同的消化质粒可以用于任何给定的 sRNA。我们相信这种方法代表了对最先进方法的重大技术改进，以促进 sRNA 生物学功能方面的表征。