Unraveling the Kinetics of the 10-23 RNA-Cleaving DNAzyme.

DNA-based enzymes, or DNAzymes, are single-stranded DNA sequences with the ability to catalyze various chemical reactions, including the cleavage of the bond between two RNA nucleotides. Lately, an increasing interest has been observed in these RNA-cleaving DNAzymes in the biosensing and therapeutic fields for signal generation and the modulation of gene expression, respectively. Additionally, multiple efforts have been made to study the effects of the reaction environment and the sequence of the catalytic core on the conversion of the substrate into product. However, most of these studies have only reported alterations of the general reaction course, but only a few have focused on how each individual reaction step is affected. In this work, we present for the first time a mathematical model that describes and predicts the reaction of the 10-23 RNA-cleaving DNAzyme. Furthermore, the model has been employed to study the effect of temperature, magnesium cations and shorter substrate-binding arms of the DNAzyme on the different kinetic rate constants, broadening the range of conditions in which the model can be exploited. In conclusion, this work depicts the prospects of such mathematical models to study and anticipate the course of a reaction given a particular environment.

DNA-only bioassay for simultaneous detection of proteins and nucleic acids.

Testing multiple biomarkers, as opposed to one, has become a preferred approach for diagnosing many heterogeneous diseases, such as cancer and infectious diseases. However, numerous technologies, including gold standard ELISA and PCR, can detect only one type of biomarker, either protein or nucleic acid (NA), respectively. In this work, we report for the first time simultaneous detection of proteins and NAs in the same solution, using solely functional NA (FNA) molecules. In particular, we combined the thrombin binding aptamer (TBA) and the 10-23 RNA-cleaving DNA enzyme (DNAzyme) in a single aptazyme molecule (Aptazyme1.15-3'), followed by extensive optimization of buffer composition, sequences and component ratios, to establish a competitive bioassay. Subsequently, to establish a multiplex bioassay, we designed a new aptazyme (Aptazyme2.20-5') by replacing the target recognition and substrate sequences within Aptazyme1.15-3'. This designing process included an in silico study, revealing the impact of the target recognition sequence on the aptazyme secondary structure and its catalytic activity. After proving the functionality of the new aptazyme in a singleplex bioassay, we demonstrated the capability of the two aptazymes to simultaneously detect thrombin and NA target, or two NA targets in a multiplex bioassay. High specificity in target detection was achieved with the limits of detection in the low nanomolar range, comparable to the singleplex bioassays. The presented results deepen the barely explored features of FNA for diagnosing multiple targets of different origins, adding an extra functionality to their catalogue.