RT-qPCR as a screening platform for mutational and small molecule impacts on structural stability of RNA tertiary structures.
RT-qPCR as a screening platform for mutational and small molecule impacts on structural stability of RNA tertiary structures.
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RT-QPCR作为突变和小分子的筛选平台对RNA三级结构的结构稳定性影响。
DOI：
10.1039/d2cb00015f
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发表时间：
2022-07-06
影响因子：
4.1
通讯作者：
Hargrove, Amanda E.
中科院分区：
文献类型：
--
作者：
Zafferani, Martina;Muralidharan, Dhanasheel;Montalvan, Nadeska, I;Hargrove, Amanda E.
RNA三级结构的发现和表征呈指数增长，突出了它们在各种人类疾病中的积极作用，但它们的相互作用组和特定功能仍然未知。小分子提供了解码这些细胞作用和开发治疗方法的机会，但是很少有靶向生物相关RNA三级结构的小分子的例子。虽然RNA三螺旋是特别有吸引力的靶标，但是三螺旋调节剂的发现由于小分子亲和力和对结构调节的作用之间缺乏相关性而受到阻碍，从而限制了基于亲和力的筛选作为主要过滤方法的实用性。为了应对这一挑战，我们开发了一个高通量RT-qPCR筛选平台，该平台报告了突变和添加剂（如小分子）对三螺旋稳定性的影响。使用致癌长非编码RNA MALAT 1的3′端作为概念验证，我们证明了两步法和一锅法的适用性，以评估突变和小分子对三螺旋稳定性的影响。我们通过将其应用于SARS-CoV-2假结，证明了该测定法对不同RNA三级结构的适应性，该假结是一种关键的病毒RNA结构，最近被确定为开发抗病毒药物的有吸引力的治疗靶点。采用功能性高通量检测作为初步筛选将显著加快发现调节RNA结构的结构景观的探针，从而有助于深入了解这些普遍存在的结构的作用。RT-qPCR可用作小分子筛选平台，以读取小分子对各种RNA靶标的结构稳定性的影响。
The exponential increase in the discovery and characterization of RNA tertiary structures has highlighted their active role in a variety of human diseases, yet often their interactome and specific function remain unknown. Small molecules offer opportunities to both decode these cellular roles and develop therapeutics, however there are few examples of small molecules that target biologically relevant RNA tertiary structures. While RNA triple helices are a particularly attractive target, discovery of triple helix modulators has been hindered by the lack of correlation between small molecule affinity and effect on structural modulation, thereby limiting the utility of affinity-based screening as a primary filtering method. To address this challenge, we developed a high-throughput RT-qPCR screening platform that reports on the effect of mutations and additives, such as small molecules, on the stability of triple helices. Using the 3′-end of the oncogenic long non-coding RNA MALAT1 as a proof-of-concept, we demonstrated the applicability of both a two-step and a one-pot method to assess the impact of mutations and small molecules on the stability of the triple helix. We demonstrated the adaptability of the assay to diverse RNA tertiary structures by applying it to the SARS-CoV-2 pseudoknot, a key viral RNA structure recently identified as an attractive therapeutic target for the development of antivirals. Employment of a functional high-throughput assay as a primary screen will significantly expedite the discovery of probes that modulate the structural landscape of RNA structures and, consequently, help gain insight into the roles of these pervasive structures. RT-qPCR can be harnessed as a small molecule screening platform to read out the effect of small molecules on the structural stability of a variety of RNA targets.
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影响因子：
4.8
作者：
Firth AE;Chung BY;Fleeton MN;Atkins JF
通讯作者：
Atkins JF
DOI：
10.1073/pnas.1219988110
发表时间：
2013-04-02
影响因子：
11.1
作者：
Hajdin, Christine E.;Bellaousov, Stanislav;Weeks, Kevin M.
通讯作者：
Weeks, Kevin M.
影响因子：
16.6
作者：
Donlic, Anita;Morgan, Brittany S.;Hargrove, Amanda E.
通讯作者：
Hargrove, Amanda E.
DOI：
10.1099/vir.0.042499-0
发表时间：
2012-07
期刊：
The Journal of general virology
影响因子：
--
作者：
Firth AE;Brierley I
通讯作者：
Brierley I
DOI：
10.1261/rna.078825.121
发表时间：
2022-03
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Jones CP;Ferré-D'Amaré AR
通讯作者：
Ferré-D'Amaré AR