| dc.description.abstract | Sodium is one of the most ubiquitous metal ions in both intracellular and extracellular fluids. Many fluorescent sensors have been designed to measure Na+ concentrations. However, Na+ binding to biomolecules such as DNA has long been considered to be non-specific. In the past few years, RNA-cleaving DNAzymes have emerged as promising tools for detecting Na+ due to their metal-specific activity. DNAzymes are DNA-based catalysts which require specific metal ions as cofactors for their catalytic activity. The initial goal of this research is to select DNAzymes that require Co(NH3)63+ as an intended cofactor through in vitro selection. However, new mutants of a previously reported Na+-specific DNAzyme were obtained instead.
An in vitro selection was preformed following a standard protocol using Co(NH3)63+ as the intended cofactor. After 6 rounds of selection in pH 6 buffer, an active sequence was successfully enriched and isolated. However, this sequence named CoH1 shows catalytic activity in the presence of Na+, instead of Co(NH3)63+. The secondary structure prediction revealed a well-defined Na+ binding domain in its catalytic core, which explained the Na+-dependent activity. After a careful comparison, the structure of CoH1 was found to be highly similar to a previously reported Na+-dependent DNAzyme, NaA43. However, two nucleotides in NaA43 that are known to be critical for its activity were mutated to different bases in CoH1. Indeed, further mutation studies indicated that any changes to these mutated positions may completely abolish the activity of CoH1.
As a new mutant of NaA43, CoH1 exhibited novel catalytic activity. With 10 mM Na+, CoH1 displays a fast cleavage rate of ~0.07 min-1, which is ~3.5-fold higher than NaA43. At pH 6, CoH1 has a stronger Na+-binding affinity with a Kd value of 4.3 ± 0.6 mM Na+, suggesting a great potential in Na+ detection at low concentrations. Based on our results, pH is important for distinguishing CoH1 from NaA43. Overall, CoH1 displays higher cleavage activity at pH below ~6.5, while NaA43 is more active at higher pH.
In addition, 2-aminopurine (2AP) was used as a fluorescence probe in converting the CoH1 DNAzyme into a folding-based Na+ sensor. 2AP is a fluorescent adenine analog whose fluorescence is strongly dependent on its local base stacking environment. By introducing a 2AP in the substrate strand, binding of Na+ induces ~80% signal enhancement. The Na+ sensor was demonstrated to be highly sensitive (a detection limit of 3.0 mM Na+) and selective over other monovalent ions. The 2AP probes also revealed the Na+-induced folding of the DNAzyme and provided important insights to the reaction mechanism. | en |
