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Fluorescent Oligonucleotides with Bis(prop-2-yn-1-yloxy)butane-1,3-diol Scaffold Rapidly Detect Disease-Associated Nucleic Acids.
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2019-12-04 , DOI: 10.1021/acs.bioconjchem.9b00746 Maria Taskova 1 , Kira Astakhova 1
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2019-12-04 , DOI: 10.1021/acs.bioconjchem.9b00746 Maria Taskova 1 , Kira Astakhova 1
Affiliation
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Biomedical research and clinical work demand rapid and reliable detection of disease-associated nucleic acids. Fluorescent oligonucleotides that bind precisely, and sense target DNA or RNA, are useful tools for simple hybridization-based assays. Although a plethora of oligonucleotide modifications are reported in the literature, they often result in poor coupling yields and are very expensive. We describe the synthesis of a new bisalkyne butane-1,3-diol scaffold and its efficient coupling into oligonucleotide sequences. We hypothesized that covalent attachment of multiple (2/4) fluorescent groups to the scaffold within oncogene-specific oligonucleotides could lead to beneficial detection of target DNA. To test this, we post-synthetically conjugated the oligonucleotides with azide-derivative dyes (2/4 per sequence): perylene, 5JOE, and (phenylethynyl)pyrene. We investigated the biophysical and photophysical properties of the oligonucleotide-dye conjugates and confirmed a "light up" fluorescent sensing mechanism of the probes upon target binding. However, fluorescence of the probes was not sensitive to mismatches. Nevertheless, "clicked" probes showed a high specificity of binding to complementary target, with the difference in Tm over 10 °C for matched vs mismatched duplex. When applied together, the mismatch-induced difference in temperature melting and fluorescence-based discrimination of the target-bound vs single-stranded probe state allowed us to apply the perylene conjugates to detect mutations in human oncogenes. Due to the beneficial target binding properties of the perylene labeled probes, along with the high fluorescence intensity of probe:target duplexes, human oncogenes could be detected in a convenient and fast (2.5 h) bead-based assay.
中文翻译：
具有双（prop-2-yn-1-yloxy）butane-1,3-diol支架的荧光寡核苷酸可快速检测与疾病相关的核酸。
生物医学研究和临床工作要求对疾病相关核酸进行快速可靠的检测。精确结合并感应目标DNA或RNA的荧光寡核苷酸是用于基于杂交的简单测定的有用工具。尽管文献中报道了过多的寡核苷酸修饰，但是它们经常导致较差的偶联产率并且非常昂贵。我们描述了一种新型的双炔炔-1,3-二醇支架的合成及其有效的偶联成寡核苷酸序列。我们假设多个（2/4）荧光基团共价连接到癌基因特异性寡核苷酸内的支架上可以导致靶DNA的有益检测。为了测试这一点，我们将寡核苷酸与叠氮化物衍生染料（每个序列2/4）合成后缀合：per，5JOE，和（苯基乙炔基）py。我们研究了寡核苷酸-染料偶联物的生物物理和光物理性质，并证实了靶标结合后探针的“点亮”荧光传感机制。但是，探针的荧光对错配不敏感。然而，“点击”探针显示出与互补靶标结合的高特异性，匹配和错配双链体的Tm超过10°C。当一起使用时，由温度匹配的不匹配引起的差异和靶结合与单链探针状态的基于荧光的区分使我们能够应用apply偶联物来检测人类致癌基因中的突变。由于the标记的探针具有有益的靶标结合特性，以及探针的高荧光强度，因此：
更新日期：2019-12-05
中文翻译：
具有双（prop-2-yn-1-yloxy）butane-1,3-diol支架的荧光寡核苷酸可快速检测与疾病相关的核酸。
生物医学研究和临床工作要求对疾病相关核酸进行快速可靠的检测。精确结合并感应目标DNA或RNA的荧光寡核苷酸是用于基于杂交的简单测定的有用工具。尽管文献中报道了过多的寡核苷酸修饰，但是它们经常导致较差的偶联产率并且非常昂贵。我们描述了一种新型的双炔炔-1,3-二醇支架的合成及其有效的偶联成寡核苷酸序列。我们假设多个（2/4）荧光基团共价连接到癌基因特异性寡核苷酸内的支架上可以导致靶DNA的有益检测。为了测试这一点，我们将寡核苷酸与叠氮化物衍生染料（每个序列2/4）合成后缀合：per，5JOE，和（苯基乙炔基）py。我们研究了寡核苷酸-染料偶联物的生物物理和光物理性质，并证实了靶标结合后探针的“点亮”荧光传感机制。但是，探针的荧光对错配不敏感。然而，“点击”探针显示出与互补靶标结合的高特异性，匹配和错配双链体的Tm超过10°C。当一起使用时，由温度匹配的不匹配引起的差异和靶结合与单链探针状态的基于荧光的区分使我们能够应用apply偶联物来检测人类致癌基因中的突变。由于the标记的探针具有有益的靶标结合特性，以及探针的高荧光强度，因此：
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