HT-SELEX-based identification of binding pre-miRNA hairpin-motif for small molecules

Abstract

Selective targeting of biologically relevant RNAs with small molecules is a long-standing challenge due to the lack of clear understanding of the binding RNA motifs for small molecules. The standard SELEX procedure allows the identification of specific RNA binders (aptamers) for the target of interest. However, more effort is needed to identify and characterize the sequence-structure motifs in the aptamers important for binding to the target. Herein, we described a strategy integrating high-throughput (HT) sequencing with conventional SELEX followed by bioinformatic analysis to identify aptamers with high binding affinity and target specificity to unravel the sequence-structure motifs of pre-miRNA, which is essential for binding to the recently developed new water-soluble small-molecule CMBL3aL. To confirm the fidelity of this approach, we investigated the binding of CMBL3aL to the identified motifs by surface plasmon resonance (SPR) spectroscopy and its potential regulatory activity on dicer-mediated cleavage of the obtained aptamers and endogenous pre-miRNAs comprising the identified motif in its hairpin loop. This new approach would significantly accelerate the identification process of binding sequence-structure motifs of pre-miRNA for the compound of interest and would contribute to increase the spectrum of biomedical application.

A TSHR-Targeting Aptamer in Monocytes Correlating with Clinical Activity in TAO

“…30 Many kinds of SELEX have been developed, based on both the target and process, including cell-SELEX, tissue-SELEX, and HT-SELEX. [31][32][33] All of these methods, however, maintain the basic logic of SELEX, including the continuous extraction of high-affinity aptamers and the discarding of low-affinity aptamers. 34 Altering the conditions of SELEX, such as temperature or binding buffer, would yield different types of aptamer.…”

计算生物学在rna研究中的应用

“…这些算法 可以使用已知的RNA-蛋白质相互作用数据作为训练集, 从中学习到特征和模式, 然后应用于预测未知的相互作用 Figure 7 Computational approaches to identify RNA-protein interaction. Experimental methods: These methods involve experimental techniques to generate high-confidence motif identification data [194,195] , RNA expression data and RNA-protein interaction data. For example, CLIP-seq is a commonly used experimental method that can identify RNA-protein interactions on a genome-wide scale.…”

Merkel cell polyomavirus regulates miR183 cluster and piR62011 in Merkel cell carcinoma

“…Mukherjee et al (2016) [14] designed a library of compounds called cyclic mismatch binding ligands (CMBLs) containing two nucleobase recognition heterocyclic moieties linked by two variable linkers in cyclophane scaffold. Total 21 CMBL molecules [21] comprising two 2-amino-1,8-naphthyridine were developed [14][15][16] to target consecutive guanines (Gs) in DNA and RNA. Expression of 41 miRNAs expressing consecutive Gs in their pre-miRNA loop regions revealed miRNA182 to be highest and thus a valid target in MCC (Fig 4C).…”