Persano Stefano, Vecchio Giuseppe, Pompa Pier Paolo
Istituto Italiano di Tecnologia, Center for Bio-Molecular Nanotechnologies@UniLe, Via Barsanti, 73010 Arnesano (Lecce), Italy.
Università del Salento, Via Provinciale Monteroni, 73100 Lecce, Italy.
Sci Rep. 2015 Apr 1;5:9558. doi: 10.1038/srep09558.
We developed a new versatile strategy that allows the detection of several classes of RNases (i.e., targeting ss- or ds-RNA, DNA/RNA hetero-hybrid or junctions) with higher sensitivity than existing assays. Our two-step approach consists of a DNA-RNA-DNA chimeric Hairpin Probe (cHP) conjugated to magnetic microparticles and containing a DNAzyme sequence in its terminal region, and molecular beacons for fluorescence signal generation. In the first step, the digestion of the RNA portion of the cHP sequences in presence of RNases leads to the release of multiple copies of the DNAzyme in solution. Then, after magnetic washing, each DNAzyme molecule elicits the catalytic cleavage of numerous molecular beacons, providing a strong amplification of the overall sensitivity of the assay. We successfully applied our approach to detect very low concentrations of RNase A, E. coli RNase I, and RNase H. Furthermore, we analyzed the effect of two antibiotics (penicillin and streptomycin) on RNase H activity, demonstrating the applicability of our strategy for the screening of inhibitors. Finally, we exploited our system to detect RNase activity directly in crude biological samples (i.e., blood and saliva) and in cell culture medium, highlighting its suitability as cheap and sensitive tool for the detection of RNase levels.
我们开发了一种新的通用策略，该策略能够检测几类核糖核酸酶（即靶向单链或双链RNA、DNA/RNA杂合链或连接点），且灵敏度高于现有检测方法。我们的两步法包括与磁性微粒结合的DNA-RNA-DNA嵌合发夹探针（cHP），其末端区域含有一个脱氧核酶序列，以及用于产生荧光信号的分子信标。第一步，在核糖核酸酶存在的情况下，cHP序列的RNA部分被消化，导致溶液中释放出多个脱氧核酶拷贝。然后，经过磁性洗涤后，每个脱氧核酶分子引发众多分子信标的催化切割，从而大大增强了检测的整体灵敏度。我们成功应用该方法检测了极低浓度的核糖核酸酶A、大肠杆菌核糖核酸酶I和核糖核酸酶H。此外，我们分析了两种抗生素（青霉素和链霉素）对核糖核酸酶H活性的影响，证明了我们的策略在筛选抑制剂方面的适用性。最后，我们利用该系统直接在粗生物样品（即血液和唾液）以及细胞培养基中检测核糖核酸酶活性，突出了其作为检测核糖核酸酶水平的廉价且灵敏工具的适用性。
