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

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.