Substrate specificity-enabled terminal protection for direct quantification of circulating MicroRNA in patient serums.

Currently， reported affinity pairings still lack in diversity， and thus terminal protection relying on steric hindrance is restricted in designing nucleic acid-based analytical systems. In this work， resistance to exonuclease is testified by group modification or backbone replacement， and the 3'-phosphate group (P) reveals the strongest exonuclease I-resistant capability. Due to the substrate specificity of enzymatic catalysis， this 3'-P protection works in a "direct mode". By introducing DNA templated copper nanoparticles， an alkaline phosphatase assay is performed to confirm the 3'-P protection. To display the application of this novel terminal protection， a multifunctional DNA is designed to quantify the model circulating microRNA (hsa-miR-21-5p) in serums from different cancer patients. According to our data， hsa-miR-21-5p-correlated cancers can be evidently distinguished from non-correlated cancers. Meanwhile， the effect of chemotherapy and radiotherapy on breast cancer is evaluated from the perspective of hsa-miR-21-5p residue in serums. Since greatly reducing the limitations of DNA design， this P-induced terminal protection can be facilely integrated with other DNA manipulations， thereby constructing more advanced biosensors with improved analytical performances for clinical applications.