DNAZYMES AS A METHOD FOR NIPAH HENIPAVIRUS DETECTION

Abstract

Intro: Deoxyribozymes (Dz) are short synthetic DNA oligonucleotides that catalyze the cleavage of a phosphodiester bond between nucleotides in the presence of divalent metal ions. The use of DNAzymes in the in vitro diagnostics increases the specificity and versatility of the analysis. Methods: We took the well-studied Dz 10-23 with high catalytic activity as the basis of our system. The biosensor is divided into two fragments according to the binary probe principle (Dz1 and Dz2), which consist of target RNA binding sites, a fluorescent substrate (Fsub), and half of the Dz 10-23 catalytic center sequence. Assembly of the Dz 10-23 active center with subsequent Fsub cleavage and registration of a fluorescent signal is possible only if the target RNA is present in the sample. Findings: To assess the diagnostic potential of the biosensor, we measured FAM fluorescence in a solution containing synthetic RNA 35 nucleotides long (nip35) corresponding to the NiV target sequence, Fsub labeled with the FAM-BHQ1 and Dz_NiV pair. A mixture of Dz_NiV and Fsub was used as a control. The detection limit of the target RNA reached 5 nM, the signal development time was 30 minutes at a temperature of 37 C ̊. Discussion: The specificity of Dz_NiV was evaluated in the presence of synthetic RNAs from six other RNA viruses of similar length: Hendra, Machupo, Sabia, Junin, Guanarito, and SARS-CoV. A fluorescent signal was recorded only in the presence of nip35 in the reaction mixture. The efficiency of Dz_NiV on a long fragment was tested using a plasmid with a cloned target sequence. The site is about 700 b.p. was amplified by PCR, followed by transcription. Conclusion: It was developed the highly specific biosensor Dz_NiV for the detection of Nipah virus RNA with a sensitivity limit of 5 nM at 37 C ̊.