Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel.
Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, 66123 Saarbrücken, Germany.
Bioconjug Chem. 2020 Apr 15;31(4):1188-1198. doi: 10.1021/acs.bioconjchem.0c00130. Epub 2020 Mar 31.
Currently, there is demand for fluorescent oligonucleotide probes for diagnostic purposes. To address this necessity, we developed nucleosides containing a flexible spacer with an intercalating moiety at its end (NIC molecules). The intercalator is based on 4-hydroxybenzylidene imidazolinone (HBI), found in the Green Fluorescent Protein. We synthesized 20-mer oligonucleotides, -, incorporating the DMTr phosphorodiamidite monomer of dU, , and the corresponding dU, , monomer. - target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of and / with complementary 2'-OMe-RNA resulted in emission at 462 and 481 nm, respectively, and up to 46-fold increase in fluorescence intensity. CD and F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes (up to Δ 6 °C). Furthermore, addition of - to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement of and . The latter sensitively detected low concentrations of the target mRNA (at total RNA 30 ng/μL). These probes were photostable for 200 min. Using a dilution curve, we quantified the number of HER-2 transcripts in a cell. In conclusion, and are promising diagnostic probes for an easy, instantaneous, specific, and sensitive detection of levels of oncogenes. Importantly, the NIC concept, demonstrated here for diagnostics of breast cancer, is universal and may be applied not only in a clinical setting but also for the detection of any RNA.
目前，人们对用于诊断目的的荧光寡核苷酸探针有需求。为满足这一需求，我们开发了含有柔性间隔基和末端嵌入部分的核苷（NIC 分子）。嵌入部分基于 4-羟基亚苄基咪唑啉酮（HBI），它存在于绿色荧光蛋白中。我们合成了 20 个核苷酸的寡核苷酸， - 靶向 HER-2 mRNA 作为乳腺癌亚型的诊断标志物。与互补 2'-OMe-RNA 的杂交分别导致 462nm 和 481nm 的发射，荧光强度增加了 46 倍。CD 和 F-NMR 数据表明，HBI 和 DFHBI 荧光团作为嵌入剂结合并稳定双链体（最多 Δ 6°C）。此外， - 与过表达 HER-2 mRNA 的癌细胞中提取的总 RNA 一起添加，导致 和 的荧光强度显著增强。后者灵敏地检测到低浓度的靶 mRNA（在总 RNA 30ng/μL 时）。这些探针在 200 分钟内具有光稳定性。通过稀释曲线，我们定量了细胞中 HER-2 转录物的数量。总之， 和 是有前途的诊断探针，可用于简单、即时、特异性和灵敏地检测致癌基因的水平。重要的是，这里展示的 NIC 概念对于乳腺癌的诊断是通用的，不仅可以在临床环境中应用，也可以用于检测任何 RNA。
