Targeting Fusobacterium nucleatum: RNA-Cleaving DNAzyme Discovery, Characterization, and Biosensor Development

Abstract

Fusobacterium nucleatum is a significant human pathogen associated with a diverse number of poor health outcomes, including but not limited to oral infections, cancer, and adverse pregnancy outcomes. Detection of F. nucleatum using qPCR has demonstrated its potential as a valuable biomarker for disease; however, current methods lack simplified approaches, limiting accessibility for F. nucleatum detection. To achieve more accessible detection, functional nucleic acids such as RNA-cleaving fluorogenic DNAzymes (RFDs) offer effective alternatives for detecting bacterial targets with high analytical sensitivity and specificity. The RFD, DT4, was isolated through an in vitro selection procedure to recognize an unknown thermally stable target of F. nucleatum. This target was present in both the crude extracellular matrix (CEM) and crude intracellular matrix (CIM), had a molecular weight of 30–100 kDa, and was proteinaceous in nature. The initial LOD was 10⁷ CFU/mL for DT4; however, incorporating an additional 36 h culturing step reduced the LOD to as low as one seeding CFU. DT4 demonstrated species specific recognition of F. nucleatum subspecies nucleatum and polymorphum and functioned effectively in complex matrices such as stool and saliva, enabling a simple mix-and-read sensor with an LOD of 10⁷ CFU/mL in F. nucleatum spiked saliva. The thermal stability of the unknown target warranted further investigation, as thermal inactivation of nucleases is an attractive feature for developing nucleic acid-based biosensors. To standardize the production of the F. nucleatum target, culturing conditions were optimized, revealing a distinct production pattern: an initial slow phase, followed by linear accumulation, and a plateau corresponding to the stationary growth phase. Despite variability causing inconsistencies in unknown target yield at specific time points, the overall pattern was consistent. Leveraging DT4 as a biosensor enabled identification of the optimal harvesting phase for obtaining large, reproducible quantities of the unknown target. Subsequent efforts to identify the unknown target involved culturing large quantities of the bacterium, removing contaminants via size exclusion chromatography, and enriching and purifying the target using electrophoretic mobility shift assays. Liquid chromatography–mass spectrometry analyses identified Fusobacterium adhesin A (FadA) as a strong candidate for the thermally stable biomarker recognized by DT4. Further optimization of the DT4 through reselection efforts revealed that no variants outperformed the wildtype DT4, underscoring its inherent catalytic robustness and highlighting essential conserved nucleotides important for RNA-cleavage. To translate these findings into practical diagnostics, DT4 was engineered into an electroactive RNA-cleaving DNAzyme (e-RCD) conjugated with methylene blue for electrochemical signal readout. Initial bead-based electrochemical assays faced challenges such as e-RCD desorption, electrode fouling, and suboptimal signals. A second generation assay, modeled after successful E. coli biosensors, achieved improved performance with a detection limit of 10⁷ CFU/mL, although further optimization is necessary for clinical validation. Collectively, these advances underscore the promise of DNAzyme based biosensors as diagnostic tools for detecting F. nucleatum, with ongoing research aimed at clinical applicability to facilitate diagnosis, treatment and prognosis of disease associated with F. nucleatum.