RESUMEN
Many aptamers have been generated by SELEX to recognize spike proteins of SARS-CoV-2, some of which have been engineered into dimeric and trimeric versions for enhanced affinity for diagnostic applications. However, no studies have been conducted to compare the utilities of monomeric, dimeric and trimeric aptamers in diagnostic assays with real clinical samples to answer the question of what levels of affinity an aptamer must have for accurate clinical diagnostics. Herein, we carried out a comparative study with two monomeric aptamers MSA1 and MSA5, one dimeric aptamer and two homotrimeric aptamers constructed with MSA1 and MSA5, with affinity varying by 1000-fold. Using a colorimetric sandwich assay to analyze 48 human saliva samples, we found that the trimeric aptamer assay (Kd = ~10 pM) can identify the SARS-CoV-2 infection much more accurately than the dimeric aptamer assay (Kd = ~100 pM) and monomeric aptamer assay (Kd = ~10,000 pM). Based on the experimental data, we theoretically predict the levels of affinity an aptamer needs to possess to achieve 80-100% sensitivity and 100% specificity. The findings from this study highlight the need for deriving very high affinity aptamers to enable highly accurate detection of viral infection for future pandemics.
RESUMEN
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.