Rational Design and Experimental Analysis of Short-Oligonucleotide Substrate Specificity for Targeting Bacterial Nucleases

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.

Ultra-Sensitive and Specific Detection of S. aureus Bacterial Cultures Using an Oligonucleotide Probe Integrated in a Lateral Flow-Based Device

“…On the other hand, the TTprobe in the S. aureus supernatant shows a total shift of the peaks to the left side of the spectrum, with two clusters of peaks (blue spectrum) corresponding to the two major fragments of the cleavage, with masses compatible with the fragments Dig-5 -mCmUmCmG (2046.116 m/z) and dTmCmGmUmUmC-3 -Bio (2451.140 m/z). This fragmentation pattern implies the cleavage by a hydrolysis reaction of the phosphodiester bond at the 5 -thymidine position, being in very good agreement with our recently reported observations [28]. As expected, no fragmentation was observed for the LF-TTprobe in the S. epidermidis supernatant (bottom panel, brown spectrum).…”

Modulating On-Demand Release of Vancomycin from Implant Coatings via Chemical Modification of a Micrococcal Nuclease-Sensitive Oligonucleotide Linker

“…Building upon this exciting on-demand release platform, we aim to further tune the oligo linker cleavage kinetics by MN to accommodate the clinical needs for a rapid (e.g., eradication of bacteria introduced by implants through inadvertent skin contact during operation) or more sustained (e.g., eradication of bacteria migrating out of infected bone matrices following tissue debridement during the revision of an infected prosthesis) on-demand release of antibiotics. Modifying internucleoside linkages has been explored as an approach to render oligo nuclease resistance, possibly by interfering with the complexation among metal ions, the nuclease active site, and the phosphodiester bond . Among them, phosphorothionate (PS) modification, first developed by Eckstein in 1966 and subsequently demonstrated with nuclease resistance, has been widely used. , Here, we test the hypothesis that by chemically modifying mCmGTTmCmG with PS at various locations (Figure A,B), susceptibility of the oligo cleavage by MN and vancomycin release kinetics can be modulated.…”

Nuclease Activity as a Biomarker in Cancer Detection

“…Specifically, biosensors based on nuclease-activatable oligonucleotides have been used to detect various conditions [133] such as infections [70,134,135] and cancer [67][68][69].…”