Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets
Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets
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DOI：
10.1021/ja0041324
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发表时间：
2002-02-13
影响因子：
15
通讯作者：
Frank-Kamenetskii, MD
中科院分区：
文献类型：
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作者：
Kuhn, H;Demidov, VV;Frank-Kamenetskii, MD
分子信标是一种灵敏的荧光探针，选择性地与指定的DNA和RNA目标杂交。它们最近已成为定量实时监测单链核酸的实用工具。在这里，我们比较研究了各种这样的探针，无茎和含有茎的DNA和PNA(肽核酸)信标在含有不同浓度的氯化钠和氯化镁的Tris-Buffer溶液中的性能。我们证明了不同的分子信标对盐浓度的变化响应不同，这可能是由于它们的骨架和结构的不同。我们发现，无茎PNA信标与互补的寡脱氧核苷酸杂交迅速，并且比DNA信标对盐的变化不那么敏感，从而可以在各种条件下有效地检测核酸靶标。尽管我们发现无茎DNA信标由于背景荧光强而不适合用于诊断目的，但我们相信在分子生物物理研究中使用这些DNA和类似的RNA结构可能有助于分析单链核酸的构象灵活性。在PNA“开启器”的帮助下，分子信标被用来直接检测双链DNA(DsDNA)中选定的目标序列。发现了无茎PNA信标强烈区分互补和不匹配dsDNA靶标的情况。再加上PNA信标对盐和DNA结合/加工蛋白的不敏感性，后者的结果表明，这些探针具有在不变性和双链DNA去蛋白的情况下识别dsDNA中特定序列的强大工具的潜力。
Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl2, We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.