Applications of RNA-based molecular logic have been hampered by sequence constraints imposed on the input and output of the circuits. Here we show that the sequence constraints can be substantially reduced by appropriately encoded multi-arm junctions of single-stranded RNA structures. To conditionally activate RNA translation, we integrated multi-arm junctions, self-assembled upstream of a regulated gene and designed to unfold sequentially in response to different RNA inputs, with motifs of loop-initiated RNA activators that function independently of the sequence of the input RNAs and that reduce interference with the output gene. We used the integrated RNA system and sequence-independent input RNAs to execute two-input and three-input OR and AND logic in Escherichia coli, and designed paper-based cell-free colourimetric assays that accurately identified two human immunodeficiency virus (HIV) subtypes (by executing OR logic) in amplified synthetic HIV RNA as well as severe acute respiratory syndrome coronavirus-2 (via two-input AND logic) in amplified RNA from saliva samples. The sequence-independent molecular logic enabled by the integration of multi-arm junction RNAs with motifs for loop-initiated RNA activators may be broadly applicable in biotechnology.

基于 RNA 的分子逻辑的应用受到对电路输入和输出施加的序列限制的阻碍。在这里，我们表明可以通过适当编码的单链 RNA 结构的多臂连接大大减少序列限制。为了有条件地激活 RNA 翻译，我们整合了多臂连接，在受调节基因的上游自组装，并设计为响应不同的 RNA 输入而顺序展开，具有独立于输入序列的环启动 RNA 激活因子的基序RNA 和减少对输出基因的干扰。我们使用集成 RNA 系统和序列无关的输入 RNA 在大肠杆菌中执行两输入和三输入 OR 和 AND 逻辑，并设计了基于纸的无细胞比色法，准确识别了扩增合成 HIV RNA 中的两种人类免疫缺陷病毒 (HIV) 亚型（通过执行或逻辑）以及严重急性呼吸系统综合症冠状病毒-2（通过双输入与逻辑） ) 从唾液样本中扩增 RNA。通过将多臂连接 RNA 与环启动 RNA 激活剂的基序整合而实现的与序列无关的分子逻辑可能广泛应用于生物技术。