Abstract:

Ag10c is a recently reported RNA-cleavingDNAzyme obtained from in vitro selection. Its cleavageactivity selectively requires Ag+ions, and thus it has been usedas a sensor for Ag+detection. However, the previous selectionyielded very limited information regarding its sequencerequirement, since only∼0.1% of the population in thefinallibrary were related to Ag10c and most other sequences wereinactive. In this work, we performed a reselection byrandomizing the 19 important nucleotides in Ag10c in sucha way that a purine has an equal chance of being A or G, whereas a pyrimidine has an equal chance of being T or C. The round3 library of the reselection was carefully analyzed and a statistic understanding of the relative importance of each nucleotide wasobtained. At the same time, a more active mutant was identified, containing two mutated nucleotides. Further analysis indicatednew base pairs leading to an enzyme with smaller catalytic loops but with∼200% activity of the original Ag10c, and alsoexcellent selectivity for Ag+. Therefore, a more active mutant of Ag10c was obtained and further truncations were successfullyperformed, which might be better candidates for developing new biosensors for silver. A deeper biochemical understanding wasalso obtained using this reselection method.