A highly specific sodium aptamer probed by 2-aminopurine for robust Na⁺sensing

Abstract

Sodium is one of the most abundant metals in the environment and in biology, playing critical ecological and physiological roles. Na+ is also the most common buffer salt for nucleic acids research, while its specific interaction with DNA has yet to be fully studied. Herein, we probe a highly selective and robust Na+ aptamer using 2-aminopurine (2AP), a fluorescent adenine analog. This aptamer has two DNA strands derived from the Ce13d DNAzyme. By introducing a 2AP at the cleavage site of the substrate strand, Na+ induces ∼40% fluorescence increase. The signaling is improved by a series of rational mutations, reaching >600% with the C10A20 double mutant. This fluorescence enhancement suggests relaxed base stacking near the 2AP label upon Na+ binding. By replacing a non-conserved adenine in the enzyme strand by 2AP, Na+-dependent fluorescence quenching is observed, suggesting that the enzyme loop folds into a more compact structure upon Na+ binding. The fluorescence changes allow for Na+ detection. With an optimized sequence, a detection limit of 0.4 mM Na+ is achieved, reaching saturated signal in less than 10 s. The sensor response is insensitive to ionic strength, which is critical for Na+ detection.

Theranostic DNAzymes

“…Through detailed characterization, NaA43 and Ce13d contain the same Na + binding aptamer in their structures, and their identical sequence is important for Na + binding 45, 46. The Na + binding aptamer in the Ce13d was further dissected by various biological and spectroscopic techniques, by which the aptamer was found to have a K d value of 20-40 mM Na + 47, 48.…”

From general base to general acid catalysis in a sodium-specific DNAzyme by a guanine-to-adenine mutation

“…The fluorescence of 2AP is highly sensitive to its nearby base stacking environment (43). Thus, 2AP has been frequently used to probe local folding of DNAzymes and ribozymes (44–46). We then substituted A22 by 2AP (Figure 5A).…”

Catalytic Nucleic Acids: Biochemistry, Chemical Biology, Biosensors, and Nanotechnology

“…An interesting example is the Na + -binding loop seen in the NaA43 and Ce13d DNAzymes. By introducing a 2AP-modification to the cleavage site, the Na + -induced folding enhances the fluorescence indicating a relaxed stacking environment near the 2AP base ( Figure 11D) (Zhou et al, 2016a). This method also indicates that K + can misfold and inactivate the DNAzyme, thus explaining the very high selectivity for Na + (He et al, 2018).…”

“…The most popular method is fluorescence spectroscopy owing to its high sensitivity, rich molecular information, and simplicity in instrumentation. For example, the global or local folding of many RNA-cleaving DNAzymes have been explored by fluorescence techniques such as 2-aminopurine (2AP) modification (Zhou et al, 2016a), fluorescence resonance energy transfer (FRET) (Kim et al, 2007), and sensitized Tb 3+ luminescence (Kim et al, 2008). An interesting example is the Na + -binding loop seen in the NaA43 and Ce13d DNAzymes.…”