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

Junyao Li,Zhaoyin Wang,Zhihui Dai,Wenxin Fu

doi:10.1039/c8sc05240a

Abstract

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.

Highlights

MicroRNAs are small, endogenous, noncoding RNAs found in diverse organisms, and play key roles in cell development, proliferation, differentiation and apoptosis.[1,2,3,4] Recent evidence has shown that miRNAs can repress the expression of important cancer-related genes, being considered as new biomarkers.[5,6,7] In particular, some miRNAs designated as circulating miRNAs exist in various biological uids like serum and plasma,[8] exhibiting superiority in providing abundant information for diagnosis and treatment of cancer
Reported affinity pairings still lack in diversity, and terminal protection relying on steric hindrance is restricted in designing nucleic acid-based analytical systems
By comparison of the diversities of uorescence intensity, it is found that 30T-P exhibits the strongest resistance to exonuclease I (exo I), suggesting that 30-P can effectively protect DNA from digestion of exo I

Summary

Introduction

MicroRNAs (miRNAs) are small, endogenous, noncoding RNAs found in diverse organisms, and play key roles in cell development, proliferation, differentiation and apoptosis.[1,2,3,4] Recent evidence has shown that miRNAs can repress the expression of important cancer-related genes, being considered as new biomarkers.[5,6,7] In particular, some miRNAs designated as circulating miRNAs (cmiRNAs) exist in various biological uids like serum and plasma,[8] exhibiting superiority in providing abundant information for diagnosis and treatment of cancer. 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. In combination with the simplicity of phosphorylation, effectiveness of exo I-resistant capability, and limited in uences on other DNA sequences, modi cation of 30-P is an ideal way to realize direct terminal protection.

Results

Conclusion