Shujun Wang


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Selection of a metal ligand modified DNAzyme for detecting Ni2+
Wei Ren, Po‐Jung Jimmy Huang, Donatien de Rochambeau, Woohyun J. Moon, Jinyi Zhang, Mingsheng Lyu, Shujun Wang, Hanadi F. Sleiman, Juewen Liu
Biosensors and Bioelectronics, Volume 165

Abstract Nickel is a highly important metal, and the detection of Ni2+ using biosensors is a long-stand analytical challenge. DNA has been widely used for metal detection, although no DNA-based sensors were reported for Ni2+. DNAzymes are DNA-based catalysts, and they recruit metal ions for catalysis. In this work, in vitro selection of RNA-cleaving DNAzymes was carried out using a library containing a region of 50 random nucleotides in the presence of Ni2+. To increase Ni2+ binding, a glycyl–histidine-functionalized tertiary amine moiety was inserted at the cleavage junction. A representative DNAzyme named Ni03 showed a high cleavage yield with Ni2+ and it was further studied. After truncation, the optimal sequence of Ni03l could bind one Ni2+ or two Co2+ for catalysis, while other metal ions were inactive. Its cleavage rates for 100 μM Ni2+ reached 0.63 h−1 at pH 8.0. A catalytic beacon biosensor was designed by labeling a fluorophore and a quencher on the Ni03l DNAzyme. Fluorescence enhancement was observed in the presence of Ni2+ with a detection limit of 12.9 μM. The sensor was also tested in spiked Lake Ontario water achieving a similar sensitivity. This is another example of using single-site modified DNAzyme for sensing transition metal ions.


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Reselection Yielding a Smaller and More Active Silver-Specific DNAzyme
Lide Gu, Runjhun Saran, Wanli Yan, Po‐Jung Jimmy Huang, Shujun Wang, Mingsheng Lyu, Juewen Liu
ACS Omega, Volume 3, Issue 11

Ag10c is a recently reported RNA-cleaving DNAzyme obtained from in vitro selection. Its cleavage activity selectively requires Ag+ ions, and thus it has been used as a sensor for Ag+ detection. However, the previous selection yielded very limited information regarding its sequence requirement, since only ∼0.1% of the population in the final library were related to Ag10c and most other sequences were inactive. In this work, we performed a reselection by randomizing the 19 important nucleotides in Ag10c in such a 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 round 3 library of the reselection was carefully analyzed and a statistic understanding of the relative importance of each nucleotide was obtained. At the same time, a more active mutant was identified, containing two mutated nucleotides. Further analysis indicated new base pairs leading to an enzyme with smaller catalytic loops but with ∼200% activity of the original Ag10c, and also excellent selectivity for Ag+. Therefore, a more active mutant of Ag10c was obtained and further truncations were successfully performed, which might be better candidates for developing new biosensors for silver. A deeper biochemical understanding was also obtained using this reselection method.