"Bind, cleave and leave": multiple turnover catalysis of RNA cleavage by bulge-loop inducing supramolecular conjugates.
"Bind, cleave and leave": multiple turnover catalysis of RNA cleavage by bulge-loop inducing supramolecular conjugates.
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DOI：
10.1093/nar/gkab1273
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发表时间：
2022-01-25
影响因子：
14.9
通讯作者：
Bichenkova EV
中科院分区：
文献类型：
--
作者：
Amirloo B;Staroseletz Y;Yousaf S;Clarke DJ;Brown T;Aojula H;Zenkova MA;Bichenkova EV
反义序列特异性敲低致病RNA为寻找新的治疗方法提供了机会。然而，为了获得预期的治疗效果，多重翻转催化对于使许多新出现的RNA序列失活至关重要，而这很难在不牺牲切割序列特异性的情况下实现。在这里，将两个或三个催化肽工程到反义寡核苷酸的大环诱导分子框架中，实现了靶RNA的催化转换。不同的超分子构型表明，随着催化胍基团数量的增加，与催化剂进行序列特异性杂交时RNA主干的切割加速，在24小时内几乎完全拆除目标RNA。在膨胀环内和周围不同位置的多个序列特异性切割促进了催化剂的释放，从而促进了后续至少10个RNA底物拷贝的攻击。因此，仅递送少量催化分子就足以维持典型RNA拷贝数的降低。我们已经开发了荧光分析和动力学模拟工具，以表征不同靶点和催化剂的有限可用性如何在很大程度上限制了催化反应的进展，并告知如何进一步加速对较短的线性和较大的rna的催化破坏。在RNA切割过程中，“智能”共轭物携带多个切割基团，从不同方向攻击诱导的凸环，从而催化了多个底物的转换。靶区的多次切割导致RNA切割产物的大小减小，从而促进RNA-偶联物复合物的崩溃和偶联物的释放，从而为下一次对目标RNA的攻击提供了条件。通过反应催化翻转的多个RNA拷贝的破坏在这里通过荧光监测。
Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge–loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge–loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further. Multiple substrate turnover in RNA cleavage is catalysed by ‘smart’ conjugate bearing several cleaving groups to attack the induced bulge–loops from different directions. Multiple cuts in the target region cause the reduction in size of the RNA cleavage products, thus facilitating collapse of the RNA-conjugate complex and release of the conjugate, which then becomes available for the next attack on target RNA. The destruction of multiple RNA copies through reaction catalytic turnover is monitored here by fluorescence.
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