Fraunhofer Institute for Applied Polymer Research IAP, Life Science and Bioprocesses, Potsdam, Germany.
Fraunhofer Cluster of Excellence Immune-Mediated Diseases CIMD, Frankfurt am Main, Germany.
PLoS One. 2024 Aug 16;19(8):e0308084. doi: 10.1371/journal.pone.0308084. eCollection 2024.
Fluorescence-based oligonucleotide probes have a great importance in research of molecular interactions. Molecular beacons (MBs) are special case of fluorescent probes that form a stem-loop shape, bringing together a fluorophore and quencher, thus emitting fluorescence only when hybridized to a complementary target. Here we describe a new method for the quantitation of MB hybridization based on the measurement of changes in free energy instead of the fluorescence intensity. The MB energy state can be measured by micro-fluorescence detection. The approach allowed to determine hybridization energy of the MB with target nucleotide directly from fluorescence spectra and distinguish the MB in unfolded and hybridized states. Moreover, the method enabled us to discriminate between DNA duplexes with perfect complementarity or a single-nucleotide mismatch, based on the first direct experimental prove of enthalpy-entropy compensation.
基于荧光的寡核苷酸探针在分子相互作用的研究中具有重要意义。分子信标（MBs）是荧光探针的特殊情况，形成茎环形状，将荧光团和猝灭剂聚集在一起，只有与互补靶标杂交时才会发出荧光。在这里，我们描述了一种新的基于自由能而不是荧光强度测量来定量 MB 杂交的方法。MB 的能量状态可以通过微荧光检测来测量。该方法允许我们直接从荧光光谱中确定 MB 与靶核苷酸的杂交能，并区分展开和杂交状态的 MB。此外，该方法使我们能够根据首次直接实验证明的焓熵补偿，区分具有完全互补或单个核苷酸错配的 DNA 双链。
