Abstract
An undecamer oligonucleotide probe based on a pair of deoxythymidines flanked by several modified nucleotides is a specific and highly efficient biosensor for micrococcal nuclease (MNase), an endonuclease produced by Staphylococcus aureus. Herein, the interaction mode and cleavage process on such oligonucleotide probes are identified and described for the first time. Also, we designed truncated pentamer probes as the minimum-length substrates required for specific and efficient biosensing. By means of computational (virtual docking) and experimental (ultra-performance liquid chromatography–mass spectrometry and matrix-assisted laser desorption ionization time-of-flight) techniques, we perform a sequence/structure–activity relationship analysis, propose a catalytically active substrate–enzyme complex, and elucidate a novel two-step phosphodiester bond hydrolysis mechanism, identifying the cleavage sites and detecting and quantifying the resulting probe fragments. Our results unravel a picture of both the enzyme–biosensor complex and a two-step cleavage/biosensing mechanism, key to the rational oligonucleotide design process.
Highlights
Nucleic acids have been proven useful recognition molecules for the development of several diagnostic strategies, taking advantage of their flexibility to be adapted to various transduction mechanisms, such as fluorescence, electrochemistry, piezoelectric, and colorimetric mechanisms.[1]
Pathogen specificity of seven nuclease-activatable oligonucleotide probes (NAOPs) (DNA, poly A, poly T, poly AT, 2′OMe, 2′F, and fluorescence resonance energy transfer (FRET)-dTdT; see Table 1 for sequence details) was tested by an nuclease activity assays (NAAs) carrying out the well-established FRET fluorescence method described before[4] and using five different pathogen strains (S. aureus, P. mirabilis, K. pneumoniae, P. aeruginosa, and S. pneumoniae), as well as purified micrococcal nuclease (MNase) (Cf 0.1 U/μL)
We conclude that the FRET-dTdTprobe was the only NAOP digested by the S. aureus bacterial supernatant and pure MNase, with activation ratio fold (ARF) in comparison with the control buffer of 22.6(±1.3) and 29.7(±1.6), respectively
Summary
Nucleic acids have been proven useful recognition molecules for the development of several diagnostic strategies, taking advantage of their flexibility to be adapted to various transduction mechanisms, such as fluorescence, electrochemistry, piezoelectric, and colorimetric mechanisms.[1] In particular, the use of nuclease-activatable oligonucleotide probes (NAOPs) as biosensors has proven its potential as a groundbreaking solution in early diagnosis of serious infectious diseases.[2,3] The detection of bacterial infection,[4−6] malignant cells in biopsies of breast cancer,[7−9] or food contamination by pathogenic bacteria[1] has been previously reported to be feasible on the basis of their nuclease activity profile.[10−12] A key aspect in the development of any diagnostic tool based on oligonucleotide probes is the necessity of designing probes which are both efficient and specific.[13] Efficiency is directly related to the extent to which the target nuclease cleavages the synthetic substrate In this respect, the oligonucleotide probe needs to be readily cleaved by the target nuclease even when the latter is in very low concentrations, such as in the early stages of infection. We describe probe candidates with low molecular weights compared with that of previous reports[17−19] that work efficiently as biosensors
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