DNAzyme turnover experiment

A DNAzyme turnover experiment measures how much reaction product a DNAzyme forms over time relative to the amount of active catalyst. Its central distinction is between one productive event per catalyst molecule and repeated substrate processing. The original deoxyribozyme paper reported an intermolecular catalyst with repeated turnover, rather than merely stable binding to its target.^[ref]

The observable should be chemically identified product, for example a resolved cleavage product quantified from an appropriately calibrated gel electrophoresis signal. Record a time course and distinguish the catalyst-dependent contribution from initial product and substrate-only background. An altered-sequence control tests sequence dependence; it does not automatically replace a measurement of background chemistry.

For a constructed example, suppose the initial substrate concentration is $200\mathrm{nM}$ and active catalyst concentration is $5\mathrm{nM}$. Background-corrected product reaches $40\mathrm{nM}$ after 20 minutes. ThenThe first is a dimensionless cumulative count; the second is an interval-average rate. Neither is automatically the turnover number at saturating substrate. Substrate depletion, changes in active catalyst and product inhibition can make the time course nonlinear.

A rapid initial product burst followed by a slower nearly linear phase can indicate different kinetic stages. For illustration,contains a burst amplitude $A$ and later slope $v_{\mathrm{ss}}$. Such a curve motivates investigating DNAzyme product-release limitation, but does not uniquely prove that mechanism: catalyst heterogeneity or inactivation can also alter the trace. Independent perturbations and product-release observations are needed to distinguish explanations.

A background reference matters especially when the substrate can cleave chemically without the DNAzyme. The primary analysis of chemically driven RNA degradation provides the relevant distinction between baseline cleavage and catalytic enhancement.^[ref]