当前位置：
X-MOL 学术
›
Bioconjugate Chem.
›
论文详情
Our official English website, www.x-mol.net, welcomes your
feedback! (Note: you will need to create a separate account there.)
Specific, Sensitive, and Quantitative Detection of HER-2 mRNA Breast Cancer Marker by Fluorescent Light-Up Hybridization Probes.
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2020-03-25 , DOI: 10.1021/acs.bioconjchem.0c00130 Abed Saady 1 , Melissa Wojtyniak 2 , Eli Varon 3 , Verena Böttner 2 , Noa Kinor 3 , Yaron Shav-Tal 3 , Christian Ducho 2 , Bilha Fischer 1
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2020-03-25 , DOI: 10.1021/acs.bioconjchem.0c00130 Abed Saady 1 , Melissa Wojtyniak 2 , Eli Varon 3 , Verena Böttner 2 , Noa Kinor 3 , Yaron Shav-Tal 3 , Christian Ducho 2 , Bilha Fischer 1
Affiliation
|
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, ON1–ON4, incorporating the DMTr phosphorodiamidite monomer of dUHBI, 2, and the corresponding dUDFHBI, 5b, monomer. ON1–ON4 target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of ON1/ON2 and ON3/ON4 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 19F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes (up to ΔTm 6 °C). Furthermore, addition of ON1–ON4 to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement of ON3 and ON4. 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, ON3 and ON4 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.
中文翻译：
荧光灯杂交探针对HER-2 mRNA乳腺癌标志物的特异性，灵敏和定量检测。
当前，需要用于诊断目的的荧光寡核苷酸探针。为了解决这一必要性，我们开发了含有在末端带有插入部分（NIC分子）的柔性间隔基的核苷。嵌入剂基于绿色荧光蛋白中的4-羟基亚苄基咪唑啉酮（HBI）。我们合成了20聚体寡核苷酸，ON1 - ON4，结合的dU的的DMTr磷酰单体HBI，2，和相应的dU DFHBI，5b中，单体。ON1 – ON4靶向HER-2 mRNA乳腺癌标志物，用于诊断乳腺癌亚型。ON1 / ON2的杂交和带有互补2'-OMe-RNA的ON3 / ON4分别导致在462和481 nm处发射，并且荧光强度最多增加46倍。CD和19 F-NMR数据表明HBI和DFHBI荧光团可作为嵌入剂结合并稳定双链体（最高ΔT m 6°C）。此外，在从过表达HER-2 mRNA的癌细胞提取的总RNA中添加ON1 - ON4，可显着增强ON3和ON4的荧光。后者灵敏地检测到低浓度的靶mRNA（总RNA 30 ng /μL）。这些探针光稳定200分钟。使用稀释曲线，我们量化了细胞中HER-2转录本的数量。总之，ON3和ON4是有前途的诊断探针，可轻松，瞬时，特异性和灵敏地检测癌基因水平。重要的是，此处介绍的用于诊断乳腺癌的NIC概念是通用的，不仅可以应用于临床，而且可以用于检测任何RNA。
更新日期：2020-04-23
中文翻译：
荧光灯杂交探针对HER-2 mRNA乳腺癌标志物的特异性，灵敏和定量检测。
当前，需要用于诊断目的的荧光寡核苷酸探针。为了解决这一必要性，我们开发了含有在末端带有插入部分（NIC分子）的柔性间隔基的核苷。嵌入剂基于绿色荧光蛋白中的4-羟基亚苄基咪唑啉酮（HBI）。我们合成了20聚体寡核苷酸，ON1 - ON4，结合的dU的的DMTr磷酰单体HBI，2，和相应的dU DFHBI，5b中，单体。ON1 – ON4靶向HER-2 mRNA乳腺癌标志物，用于诊断乳腺癌亚型。ON1 / ON2的杂交和带有互补2'-OMe-RNA的ON3 / ON4分别导致在462和481 nm处发射，并且荧光强度最多增加46倍。CD和19 F-NMR数据表明HBI和DFHBI荧光团可作为嵌入剂结合并稳定双链体（最高ΔT m 6°C）。此外，在从过表达HER-2 mRNA的癌细胞提取的总RNA中添加ON1 - ON4，可显着增强ON3和ON4的荧光。后者灵敏地检测到低浓度的靶mRNA（总RNA 30 ng /μL）。这些探针光稳定200分钟。使用稀释曲线，我们量化了细胞中HER-2转录本的数量。总之，ON3和ON4是有前途的诊断探针，可轻松，瞬时，特异性和灵敏地检测癌基因水平。重要的是，此处介绍的用于诊断乳腺癌的NIC概念是通用的，不仅可以应用于临床，而且可以用于检测任何RNA。
京公网安备 11010802027423号