Abstract
Nowadays a variety of biosensors are widely used in different fields, including biomedical diagnostics and self-testing. Nucleic acid-based biosensors are typically applied to detect another nucleic acid, proteins, ions, and several other types of compounds. It is most promising to develop simple and effective biosensors for the use in situations where traditional methods are not available due to their complexity and laboriousness. In this project, a novel smart deoxyribozyme-based fluorescent sensor for the detection of androgen receptor mRNA was developed. It consists of several functional modules including two deoxyribozymes 10–23, an RNA-dependent split malachite green aptamer, and an oligonucleotide platform. Deoxyribozymes specifically release a 27-nucleotide RNA fragment that is readily available for the interaction with the aptamer module. This solves a problem of secondary structure in hybridization with the target sequence of full-length mRNA. It was shown that within 24 hours the proposed sensor specifically recognized both a synthetic 60-nucleotide RNA fragment (LOD is 1.4 nM of RNA fragment at 37 °C) and a full-sized mRNA molecule of the androgen receptor. The constructed sensor is easy to use, has high efficiency and selectivity for the RNA target, and can be reconstructed for the detection of various nucleic acid sequences due to its modular structure. Thus, similar biosensors may be useful for the differential diagnosis.
Highlights
The fast and precise diagnostics of diseases are one of the key factors that allow choosing the most effective method of treatment
We demonstrate an experimental model of smart deoxyribozymebased fluorescent sensor (SDFS), designed for the quick and efficient verification of human androgen receptor (AR) mRNA
We proposed the model of SDFS (Figure 1A) developed in our laboratory, which was based on the principles of Holliday junction and consisted of several functional parts: (i) two catalytically active deoxyribozymes 10–23 (Figure 1B) with different lengths of RNA-binding sites (Dz1 9 nt right/4 nt left and Dz2 9 nt right/8 nt left), which recognize 60-AR_RNA on both sides around the site of aptamer binding, (ii) split malachite green aptamer (Figure 1C), and (iii) an oligonucleotide platform
Summary
The fast and precise diagnostics of diseases are one of the key factors that allow choosing the most effective method of treatment. In the SDFS structure split MGA has two RNA-binding sequences, which are complementary to the target 60-AR_RNA, and connected with T2 and T3 strands through polythymidine linkers (Figure 1A).
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