Bifacial PNAs Destabilize MALAT1 by 3' A-Tail Displacement from the U-Rich Internal Loop.
Bifacial PNAs Destabilize MALAT1 by 3' A-Tail Displacement from the U-Rich Internal Loop.
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双面PNA通过3‘A-尾部从富铀内环移位来破坏MALAT1的稳定性。
DOI：
10.1021/acschembio.1c00575
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发表时间：
2021-08-20
影响因子：
4
通讯作者：
Bong D
中科院分区：
文献类型：
--
作者：
Miao S;Bhunia D;Devari S;Liang Y;Munyaradzi O;Rundell S;Bong D
我们在此报道了一类新的合成试剂，用于靶向MALAT1中的核表达元件（ENE）， MALAT1是一种在许多癌症中上调的长链非编码RNA。顺式作用的ENE含有一个富含u的内部环（URIL），它与MALAT1的截断的3 ' oligo-A尾部形成一个富含11个碱基uau的三联体茎，保护末端不受核酸外切酶的酶切，并大大延长转录物的寿命。双面肽核酸（bPNAs）同样通过两个尿嘧啶碱基和一个合成碱基三聚氰胺之间的碱基三重形成结合URILs。我们合成了一组由α-连接肽、异二肽和二酮哌嗪骨架组成的低分子量bPNAs，并通过oligo-A链位移和相应的外切酶敏感性评估了它们在体外的ENE结合效果。在外切酶存在的情况下，bPNA处理大大增强了降解，ENE半衰期从60 - 24小时降至6分钟。RNA消化动力学可以清楚地区分具有相似URIL亲和力的bPNAs，突出了功能分析用于评估合成RNA结合物的实用性。体外活性反映在bPNAs处理后胰腺癌（PANC-1）细胞中MALAT1表达下调50%，这与类似的ENE a尾部位移机制引发的细胞内消化一致。从PANC-1总RNA中拉下生物素化的bPNA富集了MALAT1，支持bPNA- uril选择性。总之，这些实验建立了bPNA在体外和细胞内靶向天然转录物的可行性。诸如bPNAs之类的试剂可能是研究由顺式作用聚(A)结合RNA元件稳定的转录本的有用工具。
We report herein a new class of synthetic reagents for targeting the element for nuclear expression (ENE) in MALAT1, a long noncoding RNA upregulated in many cancers. The cis-acting ENE contains a U-rich internal loop (URIL) that forms an 11 base UAU-rich triplex stem with the truncated 3′ oligo-A tail of MALAT1, protecting the terminus from exonuclease digestion and greatly extending transcript lifetime. Bifacial peptide nucleic acids (bPNAs) similarly bind URILs via base triple formation between two uracil bases and a synthetic base, melamine. We synthesized a set of low molecular weight bPNAs composed of α-linked peptide, isodipeptide, and diketopiperazine backbones and evaluated their ENE binding efficacy in vitro via oligo-A strand displacement and consequent exonuclease sensitivity. Degradation was greatly enhanced by bPNA treatment in the presence of exonucleases, with ENE half-life plunging to 6 min from >24 h. RNA digestion kinetics could clearly distinguish between bPNAs with similar URIL affinities, highlighting the utility of functional assays for evaluating synthetic RNA binders. In vitro activity was mirrored by a 50% knockdown of MALAT1 expression in pancreatic cancer (PANC-1) cells upon treatment with bPNAs, consistent with intracellular digestion triggered by a similar ENE A-tail displacement mechanism. Pulldown from PANC-1 total RNA with biotinylated bPNA enriched MALAT1 > 4000×, supportive of bPNA-URIL selectivity. Together, these experiments establish the feasibility of native transcript targeting by bPNA in both in vitro and intracellular contexts. Reagents such as bPNAs may be useful tools for the investigation of transcripts stabilized by cis-acting poly(A) binding RNA elements.
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