A Fluorogenic DNAzyme for A Thermally Stable Protein Biomarker from Fusobacterium nucleatum, a Human Bacterial Pathogen.

Fusobacterium nucleatum has been correlated to many poor human conditions including oral infections, adverse pregnancies and cancer, and thus molecular tools capable of detecting this human pathogen can be used to develop diagnostic tests for them. Using a new selection method targeting thermally stable proteins without a counter-selection step, we derived an fluorogenic RNA-cleaving DNAzyme, named RFD-FN1, that can be activated by a thermally stable protein target that is unique to F. nucleatum subspecies. High thermal stability of protein targets is a very desirable attribute for DNAzyme-based biosensing directly with biological samples because nucleases found inherently in these samples can be heat-inactivated. We further demonstrate that RFD-FN1 can function as a fluorescent sensor in both human saliva and human stool samples. The discovery of RFD-FN1 paired with a highly thermal stable protein target presents opportunities for developing simpler diagnostic tests for this important pathogen.

A Highly Specific DNA Aptamer for RNase H2 from Clostridium difficile.

Molecular recognition elements with high specificity are of great importance for the study of molecular interactions, accurate diagnostics, drug design, and personalized medicine. Herein, a highly specific DNA aptamer for RNase H2 from Clostridium difficile (C. difficile) was generated by SELEX and minimized to 40 nucleotides. The aptamer exhibits a dissociation constant (Kd) of 1.8 ± 0.5 nM and an inhibition constant (IC50) of 7.1 ± 0.6 nM for C. difficile RNase H2, both of which are 2 orders of magnitude better for the same enzyme from other control bacteria. The fluorescent version of the aptamer can distinguish C. difficile from several other control bacteria in a cell lysate assay. This work demonstrates that a ubiquitous protein like RNase H2 can still be used as the target for the development of highly specific aptamers and the combination of the protein and the aptamer can achieve the recognition specificity needed for a diagnostic test and drug development.