Double combinatorial Optimization of trans-splicing Group I Intron Ribozymes

Trans-splicing group I introns are catalytic RNAs (ribozymes) that are based on naturally self-splicing group I introns. They can be designed to splice and reprogram RNAs indicating a disease status, which is of therapeutic interest. A critical factor for trans-splicing efficiency is the sequence of the extended guide sequence (EGS) appended to the Tetrahymena ribozyme 5'-terminus. However, no high-throughput screening methods are available where optimal EGS sequences can be identified from EGS libraries because successful EGSs are not incorporated into the splice products. Here, we introduce RABADOCS, a double combinatorial selection strategy, that indirectly identifies successful EGSs from a library containing tens of millions of unique randomized EGSs. To demonstrate this method, we optimized the EGS of trans-splicing ribozymes targeting the mutation site G1849U in JAK2-V617F mRNA, which is the most common cancer driver mutation in classical BCR-ABL1-negative myeloproliferative neoplasms (MPNs). The RABADOCS procedure selected 23 EGSs, of which seven showed high in vitro splicing activity, and nine had intermediate activity. In contrast, six randomly chosen EGSs showed no detectable splicing activity. These results confirm RABADOCS' ability to identify EGSs mediating improved trans-splicing activity. We expect that RABADOCS-based optimization will significantly expand the utilization of trans-splicing ribozymes for therapeutic purposes.

反式剪接I组内含子（trans-splicing group I introns）是一类基于天然自剪接I组内含子的催化性RNA（核酶，ribozyme）。此类核酶可经设计实现对指示疾病状态的RNA进行剪接与重编程，具备治疗应用价值。影响反式剪接效率的关键因素之一，是附加于四膜虫核酶（Tetrahymena ribozyme）5'端的延伸引导序列（extended guide sequence, EGS）的序列特征。然而目前尚无高通量筛选（high-throughput screening）方法可从EGS文库中筛选得到最优EGS序列，原因在于成功发挥功能的EGS不会整合至剪接产物内。本研究提出RABADOCS——一种双重组合筛选策略（double combinatorial selection strategy），可从包含数千万条独特随机化EGS的文库中间接鉴定有效EGS。为验证该方法的有效性，研究人员针对JAK2-V617F mRNA中的G1849U突变位点设计了靶向反式剪接核酶，并对其EGS进行优化；该突变位点是经典BCR-ABL1阴性骨髓增殖性肿瘤（myeloproliferative neoplasms, MPNs）中最常见的致癌驱动突变。通过RABADOCS流程共筛选得到23条EGS，其中7条表现出较高的体外剪接活性（in vitro splicing activity），9条表现出中等活性；与之相比，随机选取的6条EGS未检测到剪接活性。上述结果证实了RABADOCS能够筛选得到可提升反式剪接活性的EGS。研究团队预期，基于RABADOCS的优化手段将大幅拓展反式剪接核酶在治疗领域的应用潜力。