DNA Nanotweezers with Hydrolytic Activity for Enzyme-Free and Sensitive Detection of Fusion Gene via Logic Operation.

Yongjie Xu,Zhishun Lu,Xiangrong Luo,Mingsong Wu,Nana Geng

doi:10.1155/2018/4178045

Abstract

Gene fusion is a molecular event occurring in cellular proliferation and differentiation, and the occurrence of irregular fusion gene results in various malignant diseases. So, sensing fusion gene with high performance is an important task for integrating individual disease information. Here, we proposed a nonenzymatic and high-throughput fluorescent assay system for the detection of fusion gene by employing DNA nanotweezers with hydrolytic activity. This tweezer was assembled by three single-stranded DNAs and engineered with sensing elements and reporting subunits. In the absence of the fusion gene, the engineered tweezer remained opened and inactive which led to no signal output. However, the addition of fusion genes would cause structure alterations of the tweezer from open to close and further DNAzyme activation with the assembly of two reporting subunits. Then, the activated DNAzyme catalyzed fluorescence substrates for signal conversion. Taking BCR/ABL fusion gene as an example, the tweezer-based assay system showed not only excellent distinguishing capability towards different input targets but also high sensitivity with a detection limit of 5.29 pM. In addition to good detection performance, this system was simple and enzyme-free, offering a powerful nanometer tool as a smart nanodevice for sensing fusion detection.

Highlights

Gene fusions are a molecular event in cancer [1,2,3]
Gene fusions are a necessary molecular event in the defense of cancer and other diseases. Such T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs), as circularized DNA elements, are formed during the fusion process that creates T- and B-cell receptors [7, 8]. eir quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output, which reflects individual immunity as hallmarks. erefore, detecting fusion gene with high sensitivity and specificity is an urgent need for clinical diagnosis
Strand C consists of two 18-base sequences which hybridize with complementary sequences at the ends of strands A and B to form two stiff arms; the hinge is formed from a four-base single-stranded region of C between the regions hybridized to strands A and B. e free ends of strand C are loaded with sequences complementary to BCR and ABL sequences, respectively, served as reorganization elements for fusion gene

Summary

Introduction

Gene fusions are a molecular event in cancer [1,2,3]. Many fusions resulting from chromosomal rearrangements are driver mutations in tumors and are currently used as biomarkers or drug targets. Conventional methods for detecting fusion gene include real-time quantitative reverse transcription PCR [9], flow cytometry [4], chromosome analysis [5], fluorescence in situ hybridization [10], and more Such methods are still timeconsuming and complicated in operation to some extent. Ese assemblies have the ability to attain cascade amplification and logic gate operation upon including catalytic [23,24,25] and logical control elements [26,27,28] and circuits [29,30,31,32] Owing to their properties of high biocompatibility, outstanding stability, low cost, and custom synthesis, DNA-based assemblies have the potential to be powerful tools for biosensing and bioanalysis. Recognition elements respond to fusion gene logically, whereas the responses regulate the “open” and “closed” states of the tweeters and further regulate the “inactive” and “active” states of the DNAzyme. e activated DNAzymes successively cleave the fluorescence substrates, enabling the intelligent and sensitive detection of fusion events without the attending of any native enzyme

Experimental Section

Results and Discussion

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Conclusions